Nucleic acid encoding a T2R taste receptor

ABSTRACT

The invention provides nucleic acid and amino acid sequences for a novel family of taste transduction G-protein coupled receptors, antibodies to such receptors, methods of detecting such nucleic acids and receptors, and methods of screening for modulators of taste transduction G-protein coupled receptors.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/544,854 filed on Aug. 20, 2009 (now U.S. Pat. No. 7,868,150), is adivisional of U.S. patent application Ser. No. 11/978,088 filed Oct. 25,2007 (now U.S. Pat. No. 7,595,166), which is a continuation of U.S.patent application Ser. No. 10/962,365 filed Oct. 7, 2004 (now U.S. Pat.No. 7,465,550), which is a continuation of U.S. patent application Ser.No. 09/510,332 filed Feb. 22, 2000 (now U.S. Pat. No. 7,244,584), whichis a continuation-in-part of U.S. patent application Ser. No. 09/393,634filed Sep. 10, 1999 (now U.S. Pat. No. 6,558,910) all of which areherein incorporated by reference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No. 5R01DC03160, awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The invention provides isolated nucleic acid and amino acid sequences oftaste cell specific G-protein coupled receptors, antibodies to suchreceptors, methods of detecting such nucleic acids and receptors, andmethods of screening for modulators of taste cell specific G-proteincoupled receptors.

BACKGROUND OF THE INVENTION

Taste transduction is one of the most sophisticated forms ofchemotransduction in animals (see, e.g., Margolskee, BioEssays15:645-650 (1993); Avenet & Lindemann, J. Membrane Biol. 112:1-8(1989)). Gustatory signaling is found throughout the animal kingdom,from simple metazoans to the most complex of vertebrates; its mainpurpose is to provide a reliable signaling response to non-volatileligands. Each of these modalities is though to be mediated by distinctsignaling pathways mediated by receptors or channels, leading toreceptor cell depolarization, generation of a receptor or actionpotential, and release of neurotransmitter at gustatory afferent neuronsynapses (see, e.g., Roper, Ann. Rev. Neurosci. 12:329-353 (1989)).

Mammals are believed to have five basic taste modalities: sweet, bitter,sour, salty, and umami (the taste of monosodium glutamate) (see, e.g.,Kawamura & Kare, Introduction to Umami: A Basic Taste (1987); Kinnamon &Cummings, Ann. Rev. Physiol. 54:715-731(1992); Lindemann, Physiol. Rev.76:718-766 (1996); Stewart et al., Am. J. Physiol. 272:1-26 (1997)).Extensive psychophysical studies in humans have reported that differentregions of the tongue display different gustatory preferences (see,e.g., Hoffmann, Menchen. Arch. Path. Anat. Physiol. 62:516-530 (1875);Bradley et al., Anatomical Record 212: 246-249 (1985); Miller & Reedy,Physiol. Behav. 47:1213-1219 (1990)). Also, numerous physiologicalstudies in animals have shown that taste receptor cells may selectivelyrespond to different tastants (see, e.g., Akabas et al., Science242:1047-1050 (1988); Gilbertson et al., J. Gen. Physiol. 100:803-24(1992); Bernhardt et al., J. Physiol. 490:325-336 (1996); Cummings etal., J. Neurophysiol. 75:1256-1263 (1996)).

In mammals, taste receptor cells are assembled into taste buds that aredistributed into different papillae in the tongue epithelium.Circumvallate papillae, found at the very back of the tongue, containhundreds (mice) to thousands (human) of taste buds and are particularlysensitive to bitter substances. Foliate papillae, localized to theposterior lateral edge of the tongue, contain dozens to hundreds oftaste buds and are particularly sensitive to sour and bitter substances.Fungiform papillae containing a single or a few taste buds are at thefront of the tongue and are thought to mediate much of the sweet tastemodality.

Each taste bud, depending on the species, contains 50-150 cells,including precursor cells, support cells, and taste receptor cells (see,e.g., Lindemann, Physiol. Rev. 76:718-766 (1996)). Receptor cells areinnervated at their base by afferent nerve endings that transmitinformation to the taste centers of the cortex through synapses in thebrain stem and thalamus. Elucidating the mechanisms of taste cellsignaling and information processing is critical for understanding thefunction, regulation, and “perception” of the sense of taste.

Although much is known about the psychophysics and physiology of tastecell function, very little is known about the molecules and pathwaysthat mediate these sensory signaling responses (reviewed by Gilbertson,Current Opin. Neurobiol. 3:532-539 (1993)). Electrophysiological studiessuggest that sour and salty tastants modulate taste cell function bydirect entry of H⁺ and Na⁺ ions through specialized membrane channels onthe apical surface of the cell. In the case of sour compounds, tastecell depolarization is hypothesized to result from H⁺ blockage of K⁺channels (see, e.g., Kinnamon et al., Proc. Nat'l Acad. Sci. USA 85:7023-7027 (1988)) or activation of pH-sensitive channels (see, e.g.,Gilbertson et al., J. Gen. Physiol. 100:803-24 (1992)); salttransduction may be partly mediated by the entry of Na⁺ viaamiloride-sensitive Na⁺ channels (see, e.g., Heck et al., Science223:403-405 (1984); Brand et al., Brain Res. 207-214 (1985); Avenet etal., Nature 331: 351-354 (1988)).

Sweet, bitter, and umami transduction are believed to be mediated byG-protein-coupled receptor (GPCR) signaling pathways (see, e.g., Striemet al., Biochem. J. 260:121-126 (1989); Chaudhari et al., J. Neuros.16:3817-3826 (1996); Wong et al., Nature 381: 796-800 (1996)).Confusingly, there are almost as many models of signaling pathways forsweet and bitter transduction as there are effector enzymes for GPCRcascades (e.g., G protein submits, cGMP phosphodiesterase, phospholipaseC, adenylate cyclase; see, e.g., Kinnamon & Margolskee, Curr. Opin.Neurobiol. 6:506-513 (1996)). However, little is known about thespecific membrane receptors involved in taste transduction, or many ofthe individual intracellular signaling molecules activated by theindividual taste transduction pathways. Identification of such moleculesis important given the numerous pharmacological and food industryapplications for bitter antagonists, sweet agonists, and othermodulators of taste.

One taste-cell specific G protein that has been identified is calledGustducin (McLaughin et al., Nature 357:563-569 (1992)). This protein isproposed to be involved in the detection of certain bitter and sweettastes since gustducin knockout Mice show decreased sensitivity to somesweet and bitter tastants (Wong et al., Nature 381:796-800 (1996)), andbecause gustducin is expressed in a significant subset of cells from alltypes of taste papillae (McLaughin et al., Nature 357:563-569 (1992)).In addition, gustducin can be activated in vitro by stimulating tastemembranes with bitter compounds, likely through the activation of bitterreceptors (Ming et al, PNAS 95:8933-8938 (1998)).

Recently, two novel GPCRs were identified and found to be specificallyexpressed in taste cells. While these receptor proteins, called TR1 andTR2, appear to be directly involved in taste reception (Hoon et al.,Cell 96:541-551 (1999)), they are only expressed in a fraction ofmammalian taste receptor cells. For example, neither of the genes are,extensively expressed in Gustducin-expressing cells. Thus, it is clearthat additional taste-involved GPCRs remain to be discovered.

Genetic studies in mammals have identified numerous loci that areinvolved in the detection of taste. For example, psychophysical tastingstudies have shown that humans can be categorized as tasters,non-tasters, and super-tasters for the bitter substance PROP(6-n-propylthiouracil), and that PROP tasting may be conferred by adominant allele, with non-tasters having two recessive alleles andtasters having at least one dominant allele (see Bartoshuk et al.,Physiol Behav 56(6):1165-71; 58:203-204 (1994)). Recently, a locusinvolved in PROP tasting has been mapped to human interval 5p15 (Reed etal., Am. J. Hum. Genet., 64(5):1478-80 (1999)). The PROP tasting genepresent at the 5p15 locus has yet to be described, however.

In addition, a number of genes involved in taste have been mapped inmice. For example, a cluster of genes involved in bitter-taste detectionhas been mapped to a region of chromosome 6 in mice (Lush et al., GenetRes. 66:167-174 (1995)).

The identification and isolation of novel taste receptors and tastesignaling molecules would allow for new methods of pharmacological andgenetic modulation of taste transduction pathways. For example, theavailability of receptor and channel molecules would permit thescreening for high affinity agonists, antagonists, inverse agonists, andmodulators of taste cell activity. Such taste modulating compounds wouldbe useful in the pharmaceutical and food industries to customize taste.In addition, such taste cell specific molecules can serve as invaluabletools in the generation of taste topographic maps that elucidate therelationship between the taste cells of the tongue and taste sensoryneurons leading to taste centers in the brain.

SUMMARY OF THE INVENTION

The present invention thus provides novel nucleic acids encoding afamily of taste-cell specific G-protein coupled receptors. These nucleicacids and the polypeptides that they encode are referred to as the “T2R”family of G-protein coupled taste receptors. These receptors are alsoreferred to as the “SF” family of G-protein coupled taste receptors.This novel family of GPCRs includes components of the taste transductionpathway. In particular, members of this family are involved in thedetection of bitter tastes.

In one aspect, the present invention provides a method for identifying acompound that modulates taste signaling in taste cells, the methodcomprising the steps of: (i) contacting a taste transduction G-proteincoupled receptor polypeptide with the compound, the polypeptidecomprising at least about 50% amino acid identity to a sequence selectedfrom the group consisting of SEQ ID NO:166, SEQ ID NO:167, SEQ IDNO:168, SEQ ID NO:169, SEQ ID NO:170, and SEQ ID NO:171; and (ii)determining the functional effect of the compound upon the polypeptide.

In another aspect, the present invention provides a method foridentifying a compound that modulates taste signaling in taste cells,the method comprising the steps of: (i) contacting a taste transductionG-protein coupled receptor polypeptide with the compound, thepolypeptide comprising greater than about 60% amino acid sequenceidentity to a sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39,SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53,SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60,SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72,SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77,SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87,SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97,SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ IDNO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125,SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ IDNO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153,SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, and SEQ IDNO:164; and (ii) determining the functional effect of the compound uponthe polypeptide.

In another aspect, the present invention provides a method foridentifying a compound that modulates taste signaling in taste cells,the method comprising the steps of: (i) contacting a polypeptidecomprising an extracellular domain or transmembrane region, orcombination thereof, of a taste transduction G-protein coupled receptorwith the compound, the extracellular domain or transmembrane regioncomprising greater than about 60% amino acid sequence identity to theextracellular domain or transmembrane region of a polypeptide comprisinga sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62,SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68,SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73,SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79,SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89,SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99,SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ IDNO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127,SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ IDNO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155,SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, and SEQ ID NO:164; and (ii)determining the functional effect of the compound upon the extracellulardomain or transmembrane region.

In one embodiment, the polypeptide has G-protein coupled receptoractivity. In another embodiment, the functional effect is a chemicaleffect. In another embodiment, the functional effect is a physicaleffect. In another embodiment, the functional effect is determined bymeasuring binding of the compound to an extracellular domain of thepolypeptide. In another embodiment, the functional effect is determinedby measuring radiolabeled GTP binding to the polypeptide. In anotherembodiment, the polypeptide is recombinant. In another embodiment, thepolypeptide comprises an extracellular domain or transmembrane region ora combination of an extracellular domain and transmembrane region thatis covalently linked to a heterologous polypeptide, forming a chimericpolypeptide. In another embodiment, the polypeptide is linked to a solidphase, either covalently non-covalently. In another embodiment, thepolypeptide is from a rat, a mouse, or a human.

In another embodiment, the polypeptide is expressed in a cell or a cellmembrane. In another embodiment, the cell is a eukaryotic cell. Inanother embodiment, the functional effect is measured by determiningchanges in the electrical activity of a cell expressing the polypeptide.In another embodiment, the functional effect of the compound upon thepolypeptide is determined by measuring changes in intracellular cAMP,cGMP, IP3, or Ca²⁺ in a cell expressing the polypeptide. In anotherembodiment, a change in intracellular Ca²⁺ in the cell is detected bydetecting FURA-2 dependent fluorescence in the cell. In anotherembodiment, the cell is a eukaryotic cell. In another embodiment, thecell is an HEK-293 cell. In another embodiment, the polypeptide is afusion protein comprising at least about 20 consecutive N-terminal aminoacids of a rhodopsin protein. In another embodiment, the rhodopsinprotein is a bovine rhodopsin. In another embodiment, the cell comprisesGα15. In another embodiment, the polypeptide is expressed in a cell, andthe polypeptide is contacted with the compound in the presence of abitter tastant, wherein a difference in the functional effect of thebitter tastant on the cell in the presence of the compound and thefunctional effect of the bitter tastant on the cell in the absence ofthe compound indicates that the compound is capable of modulating tastesignaling in taste cells.

In another embodiment, the polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119,SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ IDNO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ED NO:137, SEQID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147,SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ IDNO:158, SEQ ID NO:160, SEQ ID NO:162, and SEQ ID NO:164.

In another aspect, the present invention provides an isolated nucleicacid encoding a taste transduction G-protein coupled receptor, thereceptor comprising greater than about 50% amino acid sequence identityto a sequence selected from the group consisting of SEQ ID NO:166, SEQID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, and SEQ IDNO:171.

In another aspect, the present invention provides an isolated nucleicacid encoding a taste transduction G-protein coupled receptor, whereinthe nucleic acid is amplified by primers that selectively hybridize tothe same sequence as degenerate primer sets encoding amino acidsequences selected from the group consisting of SEQ ID NO:166, SEQ IDNO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, and SEQ ID NO:171.

In another aspect, the present invention provides an isolated nucleicacid encoding a taste transduction G-protein coupled receptor, thereceptor comprising greater than about 60% amino acid sequence identityto a sequence selected from the group consisting of SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, and SEQ ID NO:164.

In another aspect, the present invention provides an isolated nucleicacid encoding a taste transduction G-protein coupled receptor, whereinthe nucleic acid specifically hybridizes under highly stringentconditions to a nucleic acid having a nucleotide sequence selected fromthe group consisting of SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ IDNO:84, SEQ ID NO:86; SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ IDNO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ IDNO:104 SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:120,SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ IDNO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148,SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ IDNO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, and SEQ ID NO:165,but not to a nucleic acid having a nucleotide sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:57,SEQ ID NO:61, and SEQ ID NO:63.

In another aspect, the present invention provides an isolated nucleicacid encoding a taste transduction G-protein coupled receptor, thereceptor comprising greater than about 60% amino acid identity to apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83,SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93,SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103,SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ IDNO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131,SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ IDNO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:160,SEQ ID NO:162, and SEQ ID NO:164, wherein the nucleic acid selectivelyhybridizes under moderately stringent hybridization conditions to anucleotide sequence having a nucleotide sequence selected from the groupconsisting of SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84,SEQ ID NO:86; SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94,SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104SEQ ID NO:106, SEQ ED NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ IDNO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:120, SEQID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130,SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ IDNO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:157,SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, and SEQ ID NO:165 but notto a nucleic acid having a nucleotide sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ED NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ EDNO:43, SEQ ID NO:45, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:57, SEQ IDNO:61, and SEQ ID NO:63.

In another aspect, the present invention provides an isolated nucleicacid encoding an extracellular domain or transmembrane region or acombination thereof of a taste transduction G-protein coupled receptor,the extracellular domain or transmembrane region having greater thanabout 60% amino acid sequence identity to the extracellular domain ortransmembrane region of a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQID NO:111, SEQ ID NO:113, SEQ ED NO:115, SEQ ID NO:117, SEQ ID NO:119,SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ IDNO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147,SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ IDNO:158, SEQ ID NO:160, SEQ ID NO:162, and SEQ ID NO:164.

In one embodiment, the nucleic acid encodes a receptor that specificallybinds to polyclonal antibodies generated against a polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NO:77,SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87,SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97,SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ IDNO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125,SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ IDNO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153,SEQ ED NO:155, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, and SEQ IDNO:164, but not to polyclonal antibodies generated against a polypeptidehaving an amino acid sequence selected from the group consisting of SEQED NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ED NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76.

In another embodiment, the nucleic acid encodes a receptor comprising anamino acid sequence selected from the group consisting of SEQ ID NO:77,SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87,SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97,SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ IDNO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125,SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ED NO:133, SEQ IDNO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153,SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ IDNO:164, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQID NO:170, and SEQ ID NO:171.

In another embodiment, the nucleic acid comprises a nucleotide sequenceselected from the group consisting of SEQ ED NO:78, SEQ ID NO:80, SEQ IDNO:82, SEQ ID NO:84, SEQ ID NO:86; SEQ ID NO:88, SEQ ID NO:90, SEQ IDNO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ IDNO:102, SEQ ID NO:104 SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ IDNO:128, SEQ ED NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146,SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ IDNO:156, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, andSEQ ID NO:165.

In another embodiment, the nucleic acid encodes a receptor that hasG-protein coupled receptor activity. In another embodiment, the nucleicacid is from a rat or a mouse.

In another embodiment, the nucleic acid encodes an extracellular domainor transmembrane region or combination thereof linked to a heterologouspolypeptide, forming a chimeric polypeptide. In another embodiment, thenucleic acid encodes the extracellular domain of a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85,SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95,SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105,SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ IDNO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133,SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ IDNO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQID NO:153, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162,SEQ ID NO:164, SEQ ID NO:166, SEQ ID 140:167, SEQ ID NO:168, SEQ IDNO:169, SEQ ID NO:170, and SEQ ID NO:171.

In another aspect, the present invention provides an expression vectorcomprising any of the above nucleic acids. In another aspect, thepresent invention provides isolated cells comprising the expressionvector.

In another aspect, the present invention provides an isolated tastetransduction G-protein coupled receptor, the receptor comprising greaterthan about 50% amino acid sequence identity to a sequence selected fromthe group consisting of SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQID NO:169, SEQ ID NO:170, and SEQ ID NO:171.

In another aspect, the present invention provides an isolated tastetransduction G-protein coupled receptor, the receptor comprising greaterthan about 60% amino acid sequence identity to a sequence selected fromthe group consisting of SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ IDNO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ IDNO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ IDNO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121,SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ IDNO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149,SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:158, SEQ IDNO:160, SEQ ID NO:162, and SEQ ID NO:164.

In one embodiment, the receptor comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ NO:107, SEQ ID NO:109, SEQ IDNO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129,SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ IDNO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:158,SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ IDNO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, and SEQ ID NO:171.

In another embodiment, the receptor specifically binds to polyclonalantibodies generated against a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119,SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ IDNO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147,SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ IDNO:158, SEQ ID NO:160, SEQ ID NO:162, and SEQ ID NO:164, but not topolyclonal antibodies generated against a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62,SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68,SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73,SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76. In another embodiment, thereceptor has G-protein coupled receptor activity. In another embodiment,the receptor is from a rat or a mouse.

In another aspect, the present invention provides an isolatedpolypeptide comprising an extracellular domain or a transmembrane regionor a combination thereof of a taste transduction G-protein coupledreceptor, the extracellular domain or transmembrane region comprisinggreater than about 60% amino acid sequence identity to the extracellulardomain or transmembrane region of a polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, and SEQ ID NO:164.

In one embodiment, the polypeptide encodes the extracellular domain ortransmembrane region of a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119,SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ IDNO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147,SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ IDNO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, and SEQ IDNO:171. In another embodiment, the extracellular domain or transmembraneregion is covalently linked to a heterologous polypeptide, forming achimeric polypeptide.

In one aspect, the present invention provides an antibody thatselectively binds to the receptor comprising greater than about 60%amino acid sequence identity to a sequence selected from the groupconsisting of SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83,SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93,SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103,SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ IDNO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131,SEQ ED NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ IDNO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:158; SEQ ID NO:160,SEQ ID NO:162, and SEQ ID NO:164.

In another aspect, the present invention provides an expression vectorcomprising a nucleic acid encoding a taste transduction G-proteincoupled receptor, wherein the receptor is expressed in a taste cell, thereceptor comprising greater than about 60% amino acid sequence identityto a sequence selected from the group consisting of SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, and SEQ ID NO:164.

In another aspect, the present invention provides a host celltransfected with the expression vector.

In another aspect, the present invention provides an expression cassettecomprising a polynucleotide sequence that encodes a human tastetransduction G protein coupled receptor, operably linked to aheterologous promoter, wherein the receptor comprises an amino acidsequence comprising greater than about 60% amino acid sequence identityto a sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62,SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68,SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73,SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76.

In one embodiment, the receptor comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ II)NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ IDNO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, and SEQ ID NO:76.

In another aspect, the present invention provides an isolated eukaryoticcell comprising the expression cassette.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F demonstrate that Gα1.5 couples the activation of opioidreceptor and mGluR1 receptor to the release of intracellular calcium.HEK-293 cells were transiently transfected with the Gαi coupled μ opioidreceptor or the Gαq coupled mGluR1 receptor. Transfected cellscontaining Gα15 were assayed for increases in [Ca2+]i before (a, b) andafter (c, d) the addition of receptor agonists: (c) 10 μM DAMGO and (d)20 μM trans (±) 1-amino-1,3 cyclopentane dicarboxylic acid, (ACPD).Ligand- and receptor-dependent increase in [Ca2+]I were dependent onGα15 (panels e, f). Scales indicate [Ca2+]I (nM) determined from FURA-2emission ratios.

FIGS. 2A-2C show that the first 39 amino acids of bovine rhodopsineffectively targets T2Rs to the plasma membrane of HEK-293 cells.Immunofluorescence staining of non-permeabilized cells transfected withrepresentative rho-T2R fusions was detected using an anti-rhodopsin mAbB6-30.

FIGS. 3A-3L demonstrate that T2R receptors are stimulated by bittercompounds. HEK-293 cells were transfected with rho-mT2R5 (a, d, g),rho-hT2R4 (b, e, h), and rho7 mT2R8 (c, f, i). Cells expressing mT2R5were stimulated using 1.5 μM cycloheximide (d, g) and those expressinghT2R4 and mT2R8 with 1.5 mM denatonium (e, f, h, i). No increase in[Ca2+]i was observed in the absence of Gα15 (g-i); in contrast robustGα15 dependent responses were observed in the presence of tastants (df);scales indicate [Ca2+]i (nM) determined from FURA-2 emission ratios.Line traces (j-l) show the kinetics of the [Ca2+]i changes forrepresentative cells from panels (d-f); arrows indicate addition oftastants.

FIGS. 4A-4D show that mT2R5 is a taste receptor for cycloheximide. (a)HEK-293 cells expressing Gα15 and rho-mT2R5 were challenged withmultiple pulses of 2 μM cycloheximide (CYX), 3 mM 6-n-propyl thiouracil(PROP) or 5 mM denatonium (DEN); dots and horizontal bars above thetraces indicate the time and duration of tastant pulses. Cycloheximidetriggers robust receptor activation. This experiment also illustratesdesensitization to repeated stimulation or during sustained applicationof the stimulus. (b) Responses to cycloheximide are highly specific andare not observed after addition of buffer (CON) or high concentrationsof other tastants. Abbreviations and concentrations used are:cycloheximide, CYX (5 μM); atropine, ATR (5 M); brucine, BRU (5 mM);caffeic acid, CAFF (2 mM); denatonium, DEN (5 mM); epicatechin, (−) EPI(3 mM); phenyl thiocarbamide, PTC (3 mM); 6n-propyl thiouracil, PROP (10mM); saccharin, SAC (10 mM); strychnine, STR (5 mM); sucroseoctaacetate, SOA (3 mM). Columns represent the mean±s.e of at least sixindependent experiments. (c) The mT2R5 gene from taster (DBA/2-allele)and non-taster (C57BL/6-allele) strains mediate differential [Ca2+]ichanges to pulses of cycloheximide. Horizontal bars depict the time andduration of the stimulus. 200 s was allowed to elapse between stimuli toensure that cells were not desensitized due to the successiveapplication of cycloheximide. (d) Cycloheximide dose-response of mT2R5.Changes in [Ca2+]i are shown as FURA-2 (F340/F380) ratios normalized tothe response at 30 μM cycloheximide; points represent the mean±s.e. ofat least six determinations. The non-taster allele shows a markeddecrease in cycloheximide sensitivity relative to the taster allele(EC50s of ^(˜)2.3 μM versus 0.5 μM, respectively).

FIGS. 5A-5C show that hT2R4 and mT2R8 respond to denatonium. HEK-293cells expressing Gα15 were transiently transfected with hT2R4 or mT2R8receptors and [Ca2+]i was monitored as shown in FIG. 3. (a) An increasein [Ca2+]i could be induced by stimulation with denatonium but not byvarious other bitter compounds. Response profiles of (b) hT2R4 and (c)mT2R8 to a set of nine out of 55 different bitter and sweet tastants(see Experimental Procedures) are shown. CON refers to control bufferaddition, NAR to 2 mM naringin and LYS to 5 mM lysine. Otherabbreviations and concentrations are as reported in FIG. 4. The meanFURA-2 fluorescence ratio (F340/F380) before and after ligand additionwas obtained from 100 equal sized areas that included all respondingcells. The values represent the mean±s.e. of at least 6 experiments.

FIGS. 6A-6C demonstrate that cycloheximide taster and non-taster strainsexpress different alleles of mT2R5. (a) Predicted transmembrane topologyof mT2R5; amino-acid substitutions in the allele from non-taster strainsare indicated as an alternative to the wild type amino acid, separatedby a forward slash (/) (SEQ ID NO:172). The presence of only two allelesat this locus is not unexpected because the strains that share the samepolymorphisms were derived from a common founder (Beck et al., Nat Genet24:2355 (2000)). In situ hybridization showing expression of mT2R5 insubsets of cells in the circumvallate papilla of (b) a cycloheximidetaster strain (DBA/2) and (c) a non-taster strain (C57BL/6); no strainspecific differences in expression pattern were detected in taste budsfrom other regions of the oral cavity.

FIG. 7 shows that mT2R5 activates gustducin in response tocycloheximide. (a) Insect larval cell membranes containing mT2R5activate gustducin in the presence 300 μM cycloheximide but not withoutligand (control) or in the presence of 1 mM atropine, brucine, caffeine,denatonium, phenylthiocarbamide, 6-n-propyl thiouracil, quinine,saccharin, strychnine, sucrose octaacetate. (b) Cycloheximideconcentration dependence of gustducin activation by mT2R5 was fitted bysingle-site binding (Kd=14.8+0.9 μM).

FIG. 8 provides a table including nucleic acid and protein sequences fora number of human, rat, and mouse T2R family members.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides nucleic acids encoding a novel family oftaste cell specific G-protein coupled receptors. These nucleic acids andthe receptors that they encode are referred to as members of the “T2R”family of taste cell specific G protein coupled receptors. These tastecell specific GPCRs are components of the taste transduction pathway,e.g., the bitter taste transduction pathway, and are involved in thetaste detection of substances such as the bitter substances6-n-propylthiouracil (PROP), sucrose octaacetate (soa), raffinoseundecaacetate (roa), cycloheximide (cyx), denatonium, copper glycinate(Glb), and quinine (qui).

These nucleic acids provide valuable probes for the identification oftaste cells, as the nucleic acids are specifically expressed in tastecells. For example, probes for T2R polypeptides and proteins can be usedto identity taste cells present in foliate, circumvallate, and fungiformpapillae, as well as taste cells present in the geschmackstreifen andepiglottis. In particular, T2R probes are useful to identify bittersensing, gustducin expressing taste cells. They also serve as tools forthe generation of taste topographic maps that elucidate the relationshipbetween the taste cells of the tongue and taste sensory neurons leadingto taste centers in the brain. Furthermore, the nucleic acids and theproteins they encode can be used as probes to dissect taste-inducedbehaviors.

The invention also provides methods of screening for modulators, e.g.,activators, inhibitors, stimulators, enhancers, agonists, andantagonists, of these novel taste cell GPCRs. Such modulators of tastetransduction are useful for pharmacological and genetic modulation oftaste signaling pathways. These methods of screening can be used toidentify high affinity agonists and antagonists of taste cell activity.These modulatory compounds can then be used in the food andpharmaceutical industries to customize taste, for example, to decreasethe bitter taste of foods or drugs. Thus, the invention provides assaysfor taste modulation, where members of the T2R family act as direct orindirect reporter molecules for the effect of modulators on tastetransduction. GPCRs can be used in assays, e.g., to measure changes inligand binding, ion concentration, membrane potential, current flow, ionflux, transcription, signal transduction, receptor-ligand interactions,second messenger concentrations, in vitro, in vivo, and ex vivo. In oneembodiment, members of the T2R family can be used as indirect reportersvia attachment to a second reporter molecule such as green fluorescentprotein (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964(1997)). In another embodiment, T2R family members are recombinantlyexpressed in cells, and modulation of taste transduction via GPCRactivity is assayed by measuring changes in Ca²⁺ levels and otherintracellular messages such as cAMP, cGMP, and IP3.

In a preferred embodiment, a T2R polypeptide is expressed in aeukaryotic cell as a chimeric receptor with a heterologous, chaperonesequence that facilitates its maturation and targeting through thesecretory pathway. In a preferred embodiment, the heterologous sequenceis a rhodopsin sequence, such as an N-terminal fragment of a rhodopsin.Such chimeric T2R receptors can be expressed in any eukaryotic cell,such as HEK-293 cells. Preferably, the cells comprise a functional Gprotein, e.g., Gα15, that is capable of coupling the chimeric receptorto an intracellular signaling pathway or to a signaling protein such asphospholipase Cβ. Activation of such chimeric receptors in such cellscan be detected using any standard method, such as by detecting changesin intracellular calcium by detecting FURA-2 dependent fluorescence inthe cell.

Methods of assaying for modulators of taste transduction include invitro ligand binding assays using T2R polypeptides, portions thereofsuch as the extracellular domain or transmembrane region or combinationthereof, or chimeric proteins comprising one or more domains of a T2Rfamily member; oocyte or tissue culture cell T2R gene expression, orexpression of T2R fragments or fusion proteins, such as rhodopsin fusionproteins; transcriptional activation of T2R genes; phosphorylation anddephosphorylation of T2R family members; G-protein binding to GPCRs;ligand binding assays; voltage, membrane potential and conductancechanges; ion flux assays; changes in intracellular second messengerssuch as cGMP, cAMP and inositol triphosphate; changes in intracellularcalcium levels; and neurotransmitter release.

Finally, the invention provides methods of detecting T2R nucleic acidand protein expression, allowing investigation of taste transductionregulation and specific identification of taste receptor cells. T2Rfamily members also provide useful nucleic acid probes for paternity andforensic investigations. T2R genes are also useful as a nucleic acidprobe for identifying taste receptor cells, such as foliate, fungiform,circumvallate; geschmackstreifen, and epiglottis taste receptor cells,in particular bitter-taste receptive, gustducin expressing cells. T2Rreceptors can also be used to generate monoclonal and polyclonalantibodies useful for identifying taste receptor cells. Taste receptorcells can be identified using techniques such as reverse transcriptionand amplification of mRNA, isolation of total RNA or poly A⁺ RNA,northern blotting, dot blotting, in situ hybridization, RNaseprotection, S1 digestion, probing DNA microchip arrays, western blots,and the like.

The T2R genes comprise a large family of related taste cell specificG-protein coupled receptors. Within the genome, these genes are presenteither alone or within one of several gene clusters. One gene cluster,located at human genomic region 12p13, comprises at least 9 genes, and asecond cluster, located at 7q31, comprises at least 4 genes. In total,more than 50 distinct T2R family members have been identified, includingseveral putative pseudogenes. It is estimated that the human genome maycontain as many as 80-120 distinct T2R genes, encoding as many as 40-80functional human receptors.

Some of the T2R genes have been associated with previously mappedmammalian taste-specific loci. For example, the human T2R01 is locatedat human interval 5p15, precisely where the locus underlying the abilityto taste the substance PROP has previously been mapped. In addition, thehuman gene cluster found at genomic region 12p13 corresponds to a regionof mouse chromosome 6 that has been shown to contain numerousbitter-tasting genes, including sucrose octaacetate, raffinose acetate,cycloheximide, and quinine (see, e.g., Lush et al., Genet. Res.6:167-174 (1995)). These associations indicate that the T2R genes areinvolved in the taste detection of various substances, in particularbitter substances. In addition, as shown in Example 7, infra, mouse T2R5is specifically receptive to cycloheximide, and mutations in the mT2R5gene produce a Cyx phenotype. Similarly, human T2R 4 and mouse T2R8 arespecifically receptive to both denatonium and PROP).

Functionally, the T2R genes comprise a family of related seventransmembrane G-protein coupled receptors involved in tastetransduction, which interact with a G-protein to mediate taste signaltransduction (see, e.g., Fong, Cell Signal 8:217 (1996); Baldwin, Curr.Opin. Cell Biol. 6:180 (1994)). In particular, T2Rs interact in aligand-specific manner with the G protein Gustducin.

Structurally, the nucleotide sequence of T2R family members (see, e.g.,SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 23, 25, 27, 29, 31, 34,36, 38, 41, 43, 45, 52, 54, 57, 61, 63, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 157, 159, 161, 163, and 165, isolated from rats, mice,and humans) encodes a family of related polypeptides comprising anextracellular domain, seven transmembrane domains, and a cytoplasmicdomain. Related T2R family genes from other species share at least about60% nucleotide sequence identity over a region of at least about 50nucleotides in length, optionally 100, 200, 500, or more nucleotides inlength, to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 23, 25, 27,29, 31, 34, 36, 38, 41, 43, 45, 52, 54, 57, 61, 63, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 157, 159, 161, 163, or 165, or encodepolypeptides sharing at least about 60% amino acid sequence identityover an amino acid region at least about 25 amino acids in length,optionally 50 to 100 amino acids in length to SEQ ID NO:1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 22, 24, 26, 28, 30, 32, 33, 35, 37, 39, 40, 42,44, 46-51, 53, 55, 56, 58-60, 62, 64-77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,153, 155, 158, 160, 162, or 164. T2R genes are specifically expressed intaste cells.

Several consensus amino acid sequences or domains have also beenidentified that are characteristic of T2R family members. For example,T2R family members typically comprise a sequence having at least about50%, optionally 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher,identity to SEQ ID NO:166 (corresponding, e.g., to amino acid positions16-35 in SEQ ID NO:1, and to T2R transmembrane region 1), SEQ ID NO:167(corresponding, e.g., to amino acid positions 45-58 in SEQ ID NO:1, andto T2R transmembrane region 2), SEQ ID NO:168 (corresponding, e.g., toamino acid positions 89-101 in SEQ ID NO:1, and to T2R transmembraneregion 3), SEQ ID NO:169 (corresponding, e.g., to amino acid positions102-119 in SEQ ID NO:1, and to T2R transmembrane region 3), SEQ IDNO:170 (corresponding, e.g., to amino acid positions 196-209 in SEQ IDNO:1, and to T2R transmembrane region 5), or SEQ ID NO:171(corresponding, e.g., to amino acid positions 273-286 in SEQ ID NO:35,and to T2R transmembrane region 7). These conserved domains thus can beused to identify members of the T2R family, by % identity, specifichybridization or amplification, or specific binding by antibodies raisedagainst a domain.

Several T2R genes represent apparent orthologs of each other. Forexample, human T2R01 (SEQ ID NOs:1, 2), rat T2R01 (SEQ ID NOs:77, 78),and mouse T2R19 (SEQ ID NOs:141, 142), are apparent orthologs. Inaddition, rat T2R08 (SEQ ID NOs:91, 92) and mouse T2R02 (SEQ ID NOs:107,108) are about 74% identical at the amino acid sequence level, and areeach at least about 50% identical to human T2R13 (SEQ ID NOs:24, 25).Rat T2R03 (SEQ ID NOs:81, 82) and mouse T2R18 (SEQ ID NOs:139, 140) areabout 92% identical, and are each at least about 50% identical to humanT2R16 (SEQ ID NOs:30, 31). Finally, human T2R04 (SEQ ID NOs:7, 8) andmouse T2R08 (SEQ ID NOs:119, 120) are about 67% identical to each other.

The present invention also provides polymorphic variants of the T2Rproteins provided herein. For example, in the rat T2R depicted in SEQ IDNO:77: variant #1, in which an isoleucine residue is substituted for aleucine residue at amino acid position 7; and variant #2, in which analanine residue is substituted for a glycine residue at amino acidposition 20.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:79: variant #1, in which a tyrosineresidue is substituted for a phenylalanine residue at amino acidposition 2; and variant #2, in which a valine residue is substituted foran isoleucine residue at amino acid position 62.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:81: variant #1, in which a glutamineresidue is substituted for an asparagine residue at amino acid position179; and variant #2, in which a cysteine residue is substituted for amethionine residue at amino acid position 183.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:83: variant #1, in which a glycine residueis substituted for an alanine residue at amino acid position 4; andvariant #2, in which a leucine residue is substituted for an isoleucineresidue at amino acid position 63.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:85: variant #1, in which a valine residueis substituted for an isoleucine residue at amino acid position 56; andvariant #2, in which a methionine residue is substituted for a cysteineresidue at amino acid position 57.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:87: variant #1, in which an isoleucineresidue is substituted for a valine residue at amino acid position 4;and variant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 5.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:89: variant #1, in which an alanineresidue is substituted for a glycine residue at amino acid position 79;and variant #2, in which an arginine residue is substituted for a lysineresidue at amino acid position 127.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:91: variant #1, in which a leucine residueis substituted for a valine residue at amino acid position 28; andvariant #2, in which an arginine residue is substituted for a lysineresidue at amino acid position 80.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:93: variant #1, in which an arginineresidue is substituted for a lysine residue at amino acid position 75;and variant #2, in which a methionine residue is substituted for acysteine residue at amino acid position 251.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:95: variant #1, in which a threonineresidue is substituted for a serine residue at amino acid position 48;and variant #2, in which an isoleucine residue is substituted for avaline residue at amino acid position 49.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:97: variant #1, in which a glutamic acidresidue is substituted for an aspartic acid residue at amino acidposition 25; and variant #2, in which an isoleucine residue issubstituted for a leucine residue at amino acid position 100.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:99: variant #1, in which a serine residueis substituted for a threonine residue at amino acid position 4; andvariant #2, in which an isoleucine residue is substituted for a valineresidue at amino acid position 74:

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:101: variant #1, in which an asparagineresidue is substituted for a glutamine residue at amino acid position 9;and variant #2, in which a tryptophan residue is substituted for atyrosine residue at amino acid position 18.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:103: variant #1, in which a threonineresidue is substituted for a serine residue at amino acid position 26;and variant #2, in which an isoleucine residue is substituted for avaline residue at amino acid position 8.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:105: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 4;and variant #2, in which an arginine residue is substituted for a lysineresidue at amino acid position 46.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:107: variant #1, in which a threonineresidue is substituted for a serine residue at amino acid position 3;and variant #2, in which an isoleucine residue is substituted for avaline residue at amino acid position 28.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:109: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 26;and variant #2, in which an arginine residue is substituted for a lysineresidue at amino acid position 50.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:111: variant #1, in which a glycineresidue is substituted for an alanine residue at amino acid position 4;and variant #2, in which a phenylalanine residue is substituted for atryptophan residue at amino acid position 60.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:113: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 62;and variant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 244.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:115: variant #1, in which a serine residueis substituted for a threonine residue at amino acid position 3; andvariant #2, in which a lysine residue is substituted for an arginineresidue at amino acid position 123.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:117: variant #1, in which an asparagineresidue is substituted for a glutamine residue at amino acid position65; and variant #2, in which a leucine residue is substituted for anisoleucine residue at amino acid position 68.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:119: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 2;and variant #2, in which an aspartic acid residue is substituted for aglutamic acid residue at amino acid position 4.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ II) NO:121: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 16;and variant #2, in which an arginine residue is substituted for a lysineresidue at amino acid position 46.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:123: variant #1, in which a threonineresidue is substituted for a serine residue at amino acid position 9;and variant #2, in which a tryptophan residue is substituted for aphenylalanine residue at amino acid position 14.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:125: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 24;and variant #2, in which an arginine residue is substituted for a lysineresidue at amino acid position 53.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:127: variant #1, in which a phenylalanineresidue is substituted for a tryptophan residue at amino acid position51; and variant #2, in which an arginine residue is substituted for alysine residue at amino acid position 101.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:129: variant #1, in which an isoleucineresidue is substituted for a valine residue at amino acid position 4;and variant #2, in which a glycine residue is substituted for an alanineresidue at amino acid position 52.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:131: variant #1, in which an arginineresidue is substituted for a lysine residue at amino acid position 150;and variant #2, in which a leucine residue is substituted for a valineresidue at amino acid position 225.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:133: variant #1, in which a leucineresidue is substituted for an isoleucine residue at amino acid position27; and variant #2, in which a lysine residue is substituted for anarginine residue at amino acid position 127.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:135: variant #1, in which a threonineresidue is substituted for a serine residue at amino acid position 102;and variant #2, in which a glutamic acid residue is substituted for anaspartic acid residue at amino acid position 220.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:137: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 24;and variant #2, in which an arginine residue is substituted for a lysineresidue at amino acid position 45.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:139: variant #1, in which a leucineresidue is substituted for an isoleucine residue at amino acid position50; and variant #2, in which an alanine residue is substituted for aglycine residue at amino acid position 53.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:141: variant #1, in which a serine residueis substituted for a threonine residue at amino acid position 76; andvariant #2, in which an isoleucine residue is substituted for a leucineresidue at amino acid position 131.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:143: variant #1, in which an alanineresidue is substituted for a glycine residue at amino acid position 98;and variant #2, in which a phenylalanine residue is substituted for atryptophan residue at amino acid position 153.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:145: variant #1, in which a leucineresidue is substituted for an isoleucine residue at amino acid position8; and variant #2, in which a glycine residue is substituted for analanine residue at amino acid position 100.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:147: variant #1, in which a glycineresidue is substituted for an alanine residue at amino acid position 52;and variant #2, in which a valine residue is substituted for a leucineresidue at amino acid position 75.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:149: variant #1, in which a lysine residueis substituted for an arginine residue at amino acid position 44; andvariant #2, in which a leucine residue is substituted for a valineresidue at amino acid position 49.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:151: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 5;and variant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 25.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:153: variant #1, in which a glutamic acidresidue is substituted for an aspartic acid residue at amino acidposition 7; and variant #2, in which an isoleucine residue issubstituted for a leucine residue at amino acid position 60.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:155: variant #1, in which an isoleucineresidue is substituted for a valine residue at amino acid position 7;and variant #2, in which a glycine residue is substituted for an alanineresidue at amino acid position 23.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:158: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 5;and variant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 21.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:160: variant #1, in which a leucineresidue is substituted for a valine residue at amino acid position 5;and variant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 23.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:162: variant #1, in which an isoleucineresidue is substituted for a leucine residue at amino acid position 22;and variant #2, in which an alanine residue is substituted for a glycineresidue at amino acid position 34.

The present invention also provides polymorphic variants of the T2Rprotein depicted in SEQ ID NO:164: variant #1, in which a leucineresidue is substituted for an isoleucine residue at amino acid position49; and variant #2, in which an arginine residue is substituted for alysine residue at amino acid position 76.

Specific regions of the T2R nucleotide and amino acid sequences may beused to identify polymorphic variants, interspecies homologs, andalleles of T2R family members. This identification can be made in vitro,e.g., under stringent hybridization conditions or PCR (e.g., usingprimers encoding SEQ ID NOS:166-171) and sequencing, or by using thesequence information in a computer system for comparison with othernucleotide sequences. Typically, identification of polymorphic variantsand alleles of T2R family members is made by comparing an amino acidsequence of about 25 amino acids or more, e.g., 50-100 amino acids.Amino acid identity of approximately at least 60% or above, optionally65%, 70%, 75%, 80%, 85%, or 90-95% or above typically demonstrates thata protein is a polymorphic variant, interspecies homolog, or allele of aT2R family member. Sequence comparison can be performed using any of thesequence comparison algorithms discussed below. Antibodies that bindspecifically to T2R polypeptides or a conserved region thereof can alsobe used to identify alleles, interspecies homologs, and polymorphicvariants.

Polymorphic variants, interspecies homologs, and alleles of T2R genesare confirmed by examining taste cell specific expression of theputative T2R polypeptide. Typically, T2R polypeptides having an aminoacid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137; SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:160, SEQ IDNO:162, or SEQ ID NO:164 is used as a positive control in comparison tothe putative T2R protein to demonstrate the identification of apolymorphic variant or allele of the T2R family member. The polymorphicvariants, alleles and interspecies homologs are expected to retain theseven transmembrane structure of a G-protein coupled receptor.

The present invention also provides nucleotide sequences for T2Rpromoters, which can be used to drive taste cell-specific expression ofpolynucleotides, especially in gustducin expressing taste cells that arereceptive to bitter tastants.

Nucleotide and amino acid sequence information for T2R family membersmay also be used to construct models of taste cell specific polypeptidesin a computer system. These models are subsequently used to identifycompounds that can activate or inhibit T2R receptor proteins. Suchcompounds that modulate the activity of T2R family members can be usedto investigate the role of T2R genes in taste transduction.

The isolation of T2R family members provides a means for assaying forinhibitors and activators of G-protein coupled receptor tastetransduction. Biologically active T2R proteins are useful for testinginhibitors and activators of T2R as taste transducers, especially bittertaste transducers, using in vivo and in vitro assays that measure, e.g.,transcriptional activation of T2R-dependent genes; ligand binding;phosphorylation and dephosphorylation; binding to G-proteins; G-proteinactivation; regulatory molecule binding; voltage, membrane potential andconductance changes; ion flux; intracellular second messengers such ascGMP, cAMP and inositol triphosphate; intracellular calcium levels; andneurotransmitter release. Such activators and inhibitors identifiedusing T2R family members can be used to further study taste transductionand to identify specific taste agonists and antagonists. Such activatorsand inhibitors are useful as pharmaceutical and food agents forcustomizing taste, for example to decrease the bitter taste of foods orpharmaceuticals.

The present invention also provides assays, preferably high throughputassays, to identify molecules that interact with and/or modulate a T2Rpolypeptide. In numerous assays, a particular domain of a T2R familymember is used, e.g., an extracellular, transmembrane, or intracellulardomain or region. In numerous embodiments, an extracellular domain ortransmembrane region or combination thereof is bound to a solidsubstrate, and used, e.g., to isolate ligands, agonists, antagonists, orany other molecule that can bind to and/or modulate the activity of anextracellular domain or transmembrane region of a T2R polypeptide. Incertain embodiments, a domain of a T2R polypeptide, e.g., anextracellular, transmembrane, or intracellular domain, is fused to aheterologous polypeptide, thereby forming a chimeric polypeptide, e.g.,a chimeric polypeptide with G protein coupled receptor activity. Suchchimeric polypeptides are useful, e.g., in assays to identify ligands,agonists, antagonists, or other modulators of a T2R polypeptide. Inaddition, such chimeric polypeptides are useful to create novel tastereceptors with novel ligand binding specificity, modes of regulation,signal transduction pathways, or other such properties, or to createnovel taste receptors with novel combinations of ligand bindingspecificity, modes of regulation, signal transduction pathways, etc.

Methods of detecting T2R nucleic acids and expression of T2Rpolypeptides are also useful for identifying taste cells and creatingtopological maps of the tongue and the relation of tongue taste receptorcells to taste sensory neurons in the brain. In particular, methods ofdetecting T2R can be used to identify taste cells sensitive to bittertastants. Chromosome localization of the genes encoding human T2R genescan be used to identify diseases, mutations, and traits caused by andassociated with T2R family members.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

“Taste cells” include neuroepithelial cells that are organized intogroups to form taste buds of the tongue, e.g., foliate, fungiform, andcircumvallate cells (see, e.g., Roper et al., Ann. Rev. Neurosci.12:329-353 (1989)). Taste cells also include cells of the palate, andother tissues that may contain taste cells such as the esophagus and thestomach.

“T2R” refers to one or more members of a family of G-protein coupledreceptors that are expressed in taste cells such as foliate, fungiform,and circumvallate cells, as well as cells of the palate, esophagus, andstomach (see, e.g., Hoon et al., Cell 96:541-551 (1999), hereinincorporated by reference in its entirety). This family is also referredto as the “SF family” (see, e.g., U.S. Ser. No. 09/393,634). Such tastecells can be identified because they express specific molecules such asGustducin, a taste cell specific G protein, or other taste specificmolecules (McLaughin et al., Nature 357:563-569 (1992)). Taste receptorcells can also be identified on the basis of morphology (see, e.g.,Roper, supra). T2R family members have the ability to act as receptorsfor taste transduction. T2R family members are also referred to as the“GR” family, for gustatory receptor, or “SF” family.

“T2R” nucleic acids encode a family of GPCRs with seven transmembraneregions that have “G-protein coupled receptor activity,” e.g., they bindto G-proteins in response to extracellular stimuli and promoteproduction of second messengers such as IP3, cAMP, cGMP, and Ca²⁺ viastimulation of enzymes such as phospholipase C and adenylate cyclase(for a description of the structure and function of GPCRs, see, e.g.,Fong, supra, and Baldwin, supra). A dendogram providing the relationshipbetween certain T2R family members is provided as FIG. 2. These nucleicacids encode proteins that are expressed in taste cells, in particularGustducin-expressing taste cells that are responsive to bitter tastants.A single taste cell may contain many distinct T2R polypeptides.

The term “T2R” family therefore refers to polymorphic variants, alleles,mutants, and interspecies homologs that: (1) have about 60% amino acidsequence identity, optionally about 75, 80, 85, 90, or 95% amino acidsequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:160, SEQ IDNO:162, or SEQ ID NO:164 over a window of about 25 amino acids,optionally 50-100 amino acids; (2) specifically bind to antibodiesraised against an immunogen comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ IDNO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ IDNO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ IDNO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121,SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ IDNO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149,SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:158, SEQ IDNO:160, SEQ ID NO:162, and SEQ ID NO:164, and conservatively modifiedvariants thereof; (3) specifically hybridize (with a size of at leastabout 100, optionally at least about 500-1000 nucleotides) understringent hybridization conditions to a sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:45, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:57, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ IDNO:84, SEQ ID NO:86; SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ IDNO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ NO:100, SEQ ID NO:102, SEQ IDNO:104 SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:120,SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ IDNO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148,SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ IDNO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, and SEQ ID NO:165,and conservatively modified variants thereof; (4) comprise a sequence atleast about 50% identical to an amino acid sequence selected from thegroup consisting of SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ IDNO:169, SEQ ID NO:170, and SEQ ID NO:171; or (5) are amplified byprimers that specifically hybridize under stringent hybridizationconditions to the same sequence as a degenerate primer sets encoding SEQID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170,or SEQ ID NO:171.

Topologically, sensory GPCRs have an “N-terminal domain” “extracellulardomains,” a “transmembrane domain” comprising seven transmembraneregions, cytoplasmic, and extracellular loops, “cytoplasmic domains,”and a “C-terminal domain” (see, e.g., Hoon et al., Cell 96:541-551(1999); Buck & Axel, Cell 65:175-187 (1991)). These domains can bestructurally identified using methods known to those of skill in theart, such as sequence analysis programs that identify hydrophobic andhydrophilic domains (see, e.g., Stryer, Biochemistry (3^(rd) ed. 1988);see also any of a number of Internet based sequence analysis programs,such as those found at dot.imgen.bcm.tmc.edu). Such domains are usefulfor making chimeric proteins and for in vitro assays of the invention,e.g., ligand binding assays.

“Extracellular domains” therefore refers to the domains of T2Rpolypeptides that protrude from the cellular membrane and are exposed tothe extracellular face of the cell. Such domains would include the “Nterminal domain” that is exposed to the extracellular face of the cell,as well as the extracellular loops of the transmembrane domain that areexposed to the extracellular face of the cell, i.e., the loops betweentransmembrane regions 2 and 3, and between transmembrane regions 4 and5. The “N terminal domain” region starts at the N-terminus and extendsto a region close to the start of the transmembrane domain. Theseextracellular domains are useful for in vitro ligand binding assays,both soluble and solid phase. In addition, transmembrane regions,described below, can also bind ligand either in combination with theextracellular domain or alone, and are therefore also useful for invitro ligand binding assays.

“Transmembrane domain,” which comprises the seven transmembrane“regions,” refers to the domain of T2R polypeptides that lies within theplasma membrane, and may also include the corresponding cytoplasmic(intracellular) and extracellular loops, also referred to astransmembrane domain “regions.” The seven transmembrane regions andextracellular and cytoplasmic loops can be identified using standardmethods, as described in Kyte & Doolittle, J. Mol. Biol. 157:105-132(1982)), or in Stryer, supra.

“Cytoplasmic domains” refers to the domains of T2R proteins that facethe inside of the cell, e.g., the “C terminal domain” and theintracellular loops of the transmembrane domain, e.g., the intracellularloops between transmembrane regions 1 and 2, and the intracellular loopsbetween transmembrane regions 3 and 4. “C terminal domain” refers to theregion that spans the end of the last transmembrane domain and theC-terminus of the protein, and which is normally located within thecytoplasm.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains one or more T2R nucleic acids encoding one or moreT2R proteins. Such samples include, but are not limited to, tissueisolated from humans, mice, and rats, in particular, tongue, palate, andother tissues that may contain taste cells such as the esophagus and thestomach. Biological samples may also include sections of tissues such asfrozen sections taken for histological purposes. A biological sample istypically obtained from a eukaryotic organism, such as insects,protozoa, birds, fish, reptiles, and preferably a mammal such as rat,mouse, cow, dog, guinea pig, or rabbit, and most preferably a primatesuch as chimpanzees or humans.

“GPCR activity” refers to the ability of a GPCR to transduce a signal.Such activity can be measured in a heterologous cell, by coupling a GPCR(or a chimeric GPCR) to either a G-protein or promiscuous G-protein suchas Gα15, and an enzyme such as PLC, and measuring increases inintracellular calcium using (Offermans & Simon, J. Biol. Chem.270:15175-15180 (1995)). Receptor activity can be effectively measuredby recording ligand-induced changes in [Ca²⁺]_(i) using fluorescentCa²⁺-indicator dyes and fluorometric imaging. Optionally, thepolypeptides of the invention are involved in sensory transduction,optionally taste transduction in taste cells.

The phrase “functional effects” in the context of assays for testingcompounds that modulate T2R family member mediated taste transductionincludes the determination of any parameter that is indirectly ordirectly under the influence of the receptor, e.g., functional, physicaland chemical effects. It includes ligand binding, changes in ion flux,membrane potential, current flow, transcription, G-protein binding, GPCRphosphorylation or dephosphorylation, signal transduction,receptor-ligand interactions, second messenger concentrations (e.g.,cAMP, cGMP, IP3, or intracellular Ca²⁺), in vitro, in vivo, and ex vivoand also includes other physiologic effects such increases or decreasesof neurotransmitter or hormone release.

By “determining the functional effect” is meant assays for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of a T2R family member, e.g., functional, physicaland chemical effects. Such functional effects can be measured by anymeans known to those skilled in the art, e.g., changes in spectroscopiccharacteristics (e.g., fluorescence, absorbance, refractive index),hydrodynamic (e.g., shape), chromatographic, or solubility properties,patch clamping, voltage-sensitive dyes, whole cell currents,radioisotope efflux, inducible markers, oocyte T2R gene expression;tissue culture cell T2R expression; transcriptional activation of T2Rgenes; ligand binding assays; voltage, membrane potential andconductance changes; ion flux assays; changes in intracellular secondmessengers such as cAMP, cGMP, and inositol triphosphate (IP3); changesin intracellular calcium levels; neurotransmitter release, and the like.

“Inhibitors,” “activators,” and “modulators” of T2R genes or proteinsare used interchangeably to refer to inhibitory, activating, ormodulating molecules identified using in vitro and in vivo assays fortaste transduction, e.g., ligands, agonists, antagonists, and theirhomologs and mimetics. Inhibitors are compounds that, e.g., bind to,partially or totally block stimulation, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate tastetransduction, e.g., antagonists. Activators are compounds that; e.g.,bind to, stimulate, increase, open, activate, facilitate, enhanceactivation, sensitize or up regulate taste transduction, e.g., agonists.Modulators include compounds that, e.g., alter the interaction of areceptor with: extracellular proteins that bind activators or inhibitor(e.g., ebnerin and other members of the hydrophobic carrier family);G-proteins; kinases (e.g., homologs of rhodopsin kinase and betaadrenergic receptor kinases that are involved in deactivation anddesensitization of a receptor); and arrestin-like proteins, which alsodeactivate and desensitize receptors. Modulators include geneticallymodified versions of T2R family members, e.g., with altered activity, aswell as naturally occurring and synthetic ligands, antagonists,agonists, small chemical molecules and the like. Such assays forinhibitors and activators include, e.g., expressing T2R family membersin cells or cell membranes, applying putative modulator compounds, inthe presence or absence of tastants, e.g., bitter tastants, and thendetermining the functional effects on taste transduction, as describedabove. Samples or assays comprising T2R family members that are treatedwith a potential activator, inhibitor, or modulator are compared tocontrol samples without the inhibitor, activator, or modulator toexamine the extent of inhibition. Control samples (untreated withinhibitors) are assigned a relative T2R activity value of 100%.Inhibition of a T2R is achieved when the T2R activity value relative tothe control is about 80%, optionally 50% or 25-0%. Activation of a T2Ris achieved when the T2R activity value relative to the control is 110%,optionally 150%, optionally 200-500%, or 1000-3000% higher.

“Biologically active” T2R refers to a T2R having GPCR activity asdescribed above, involved in taste transduction in taste receptor cells,in particular bitter taste transduction.

The terms “isolated” “purified” or “biologically pure” refer to materialthat is substantially or essentially free from components which normallyaccompany it as found in its native state. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A protein that is the predominant species present in apreparation is substantially purified. In particular, an isolated T2Rnucleic acid is separated from open reading frames that flank the T2Rgene and encode proteins other than a T2R. The term “purified” denotesthat a nucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. Particularly, it means that the nucleic acid orprotein is at least 85% pure, optionally at least 95% pure, andoptionally at least 99% pure.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batter et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)-   (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that form a compact unit of the polypeptide and aretypically 50 to 350 amino acids long. Typical domains are made up ofsections of lesser organization such as stretches of β-sheet andα-helices. “Tertiary structure” refers to the complete three dimensionalstructure of a polypeptide monomer. “Quaternary structure” refers to thethree dimensional structure formed by the noncovalent association ofindependent tertiary units. Anisotropic terms are also known as energyterms.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins which can be madedetectable, e.g., by incorporating a radiolabel into the peptide or usedto detect antibodies specifically reactive with the peptide.

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are optionally directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences or domains that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame (i.e., 50% identity, optionally 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or higher identity over a specified region), when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Suchsequences are then said to be “substantially identical.” This definitionalso refers to the compliment of a test sequence. Optionally, theidentity exists over a region that is at least about 50 amino acids ornucleotides in length, or more preferably over a region that is 75-100amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, as described below for the BLASTN and BLASTPprograms, or alternative parameters can be designated. The sequencecomparison algorithm then calculates the percent sequence identifies forthe test sequences relative to the reference sequence, based on theprogram parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of an algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment (see, e.g., FIG.2). PILEUP uses a simplification of the progressive alignment method ofFeng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used issimilar to the method described by Higgins & Sharp, CABIOS 5:151-153(1989). The program can align up to 300 sequences, each of a maximumlength of 5,000 nucleotides or amino acids. The multiple alignmentprocedure begins with the pairwise alignment of the two most similarsequences, producing a cluster of two aligned sequences. This cluster isthen aligned to the next most related sequence or cluster of alignedsequences. Two clusters of sequences are aligned by a simple extensionof the pairwise alignment of two individual sequences. The finalalignment is achieved by a series of progressive, pairwise alignments.The program is run by designating specific sequences and their aminoacid or nucleotide coordinates for regions of sequence comparison and bydesignating the program parameters. Using PILEUP, a reference sequenceis compared to other test sequences to determine the percent sequenceidentity relationship using the following parameters: default gap weight(3.00), default gap length weight (0.10), and weighted end gaps. PILEUPcan be obtained from the GCG sequence analysis software package, e.g.,version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395 (1984)).

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences:Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, optionally 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and0.1% SDS at 65° C. Such hybridizations and wash steps can be carried outfor, e.g., 1, 2, 5, 10, 15, 30, 60, or more minutes.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. Such hybridizations and wash steps can becarried out for, e.g., 1, 2, 5, 10, 15, 30, 60, or more minutes. Apositive hybridization is at least twice background. Those of ordinaryskill will readily recognize that alternative hybridization and washconditions can be utilized to provide conditions of similar stringency.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)).Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized antibodies. Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

An “anti-T2R” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by a T2R gene, cDNA, or asubsequence thereof.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to a T2R family member from specific species such as rat, mouse,or human can be selected to obtain only those polyclonal antibodies thatare specifically immunoreactive with the T2R protein or an immunogenicportion thereof and not with other proteins, except for orthologs orpolymorphic variants and alleles of the T2R protein. This selection maybe achieved by subtracting out antibodies that cross-react with T2Rmolecules from other species or other T2R molecules. Antibodies can alsobe selected that recognize only T2R GPCR family members but not GPCRsfrom other families. A variety of immunoassay formats may be used toselect antibodies specifically immunoreactive with a particular protein.For example, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity). Typically a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background.

In one embodiment, immunogenic domains corresponding to SEQ IDNOs:166-171 can be used to raise antibodies that specifically bind topolypeptides of the T2R family.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa,HEK-293, and the like, e.g., cultured cells, explants, and cells invivo.

III. Isolation of Nucleic Acids Encoding T2R Family Members

A. General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter etal., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences EncodingT2R Family Members

In general, the nucleic acid sequences encoding T2R family members andrelated nucleic acid sequence homologs are cloned from cDNA and genomicDNA libraries by hybridization with probes, or isolated usingamplification techniques with oligonucleotide primers. For example, T2Rsequences are typically isolated from mammalian nucleic acid (genomic orcDNA) libraries by hybridizing with a nucleic acid probe, the sequenceof which can be derived from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:57, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:78, SEQ ID NO:80, SEQ IDNO:82, SEQ ID NO:84, SEQ ID NO:86; SEQ ID NO:88, SEQ ID NO:90, SEQ IDNO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ IDNO:102, SEQ ID NO:104 SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ IDNO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146,SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ IDNO:156, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, orSEQ ID NO:165. A suitable tissue from which RNA and cDNA for T2R familymembers can be isolated is tongue tissue, optionally taste bud tissuesor individual taste cells.

Amplification techniques using primers can also be used to amplify andisolate T2R sequences from DNA or RNA. For example, degenerate primersencoding the following amino acid sequences can be used to amplify asequence of a T2R gene: SEQ ID NOS: 166, 167, 168, 169, 170, or 171(see, e.g., Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual(1995)). These primers can be used, e.g., to amplify either the fulllength sequence or a probe of one to several hundred nucleotides, whichis then used to screen a mammalian library for full-length T2R clones.As described above, such primers can be used to isolate a full lengthsequence, or a probe which can then be used to isolated a full lengthsequence, e.g., from a library.

Nucleic acids encoding T2R can also be isolated from expressionlibraries using antibodies as probes. Such polyclonal or monoclonalantibodies can be raised using the sequence of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24,SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:35, SEQ ID NO:37, SEQ NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ IDNO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ IDNO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ IDNO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121,SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ IDNO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149,SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:158, SEQ IDNO:160, SEQ ID NO:162, or SEQ ID NO:164.

Polymorphic variants, alleles, and interspecies homologs that aresubstantially identical to a T2R family member can be isolated using T2Rnucleic acid probes, and oligonucleotides under stringent hybridizationconditions, by screening libraries. Alternatively, expression librariescan be used to clone T2R family members and T2R family memberpolymorphic variants, alleles, and interspecies homologs, by detectingexpressed homologs immunologically with antisera or purified antibodiesmade against a T2R polypeptide, which also recognize and selectivelybind to the T2R homolog.

To make a cDNA library, one should choose a source that is rich in T2RmRNA, e.g., tongue tissue, or isolated taste buds. The mRNA is then madeinto cDNA using reverse transcriptase, ligated into a recombinantvector, and transfected into a recombinant host for propagation,screening and cloning. Methods for making and screening cDNA librariesare well known (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983);Sambrook et al., supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

An alternative method of isolating T2R nucleic acid and its homologscombines the use of synthetic oligonucleotide primers and amplificationof an RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and 4,683,202;PCR Protocols: A Guide to Methods and Applications (Innis et al., eds,1990)). Methods such as polymerase chain reaction (PCR) and ligase chainreaction (LCR) can be used to amplify nucleic acid sequences of T2Rgenes directly from mRNA, from cDNA, from genomic libraries or cDNAlibraries. Degenerate oligonucleotides can be designed to amplify T2Rfamily member homologs using the sequences provided herein. Restrictionendonuclease sites can be incorporated into the primers. Polymerasechain reaction or other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of T2R-encoding mRNA in physiological samples,for nucleic acid sequencing, or for other purposes. Genes amplified bythe PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Gene expression of T2R family members can also be analyzed by techniquesknown in the art, e.g., reverse transcription and amplification of mRNA,isolation of total RNA or poly A⁺ RNA, northern blotting, dot blotting,in situ hybridization, RNase protection, probing DNA microchip arrays,and the like. In one embodiment, high density oligonucleotide analysistechnology (e.g., GeneChip™) is used to identify homologs andpolymorphic variants of the GPCRs of the invention. In the case wherethe homologs being identified are linked to a known disease, they can beused with GeneChip™ as a diagnostic tool in detecting the disease in abiological sample, see, e.g., Gunthand et al., AIDS Res. Hum.Retroviruses 14: 869-876 (1998); Kozal et al., Nat. Med. 2:753-759(1996); Matson et al., Anal. Biochem. 224:110-106 (1995); Lockhart etal., Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et al., Genome Res.8:435-448 (1998); Hacia et al., Nucleic Acids Res. 26:3865-3866 (1998).

Synthetic oligonucleotides can be used to construct recombinant T2Rgenes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and nonsense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the T2R nucleic acid. The specificsubsequence is then ligated into an expression vector.

The nucleic acid encoding a T2R gene is typically cloned intointermediate vectors before transformation into prokaryotic oreukaryotic cells for replication and/or expression. These intermediatevectors are typically prokaryote vectors, e.g., plasmids, or shuttlevectors.

Optionally, nucleic acids encoding chimeric proteins comprising a T2Rpolypeptide or domains thereof can be made according to standardtechniques. For example, a domain such as a ligand binding domain (e.g.,an extracellular domain alone, an extracellular domain plus atransmemberane region, or a transmembrane region alone), anextracellular domain, a transmembrane domain (e.g., one comprising up toseven transmembrane regions and corresponding extracellular andcytosolic loops), the transmembrane domain and a cytoplasmic domain, anactive site, a subunit association region, etc., can be covalentlylinked to a heterologous protein. For example, an extracellular domaincan be linked to a heterologous GPCR transmembrane domain, or aheterologous GPCR extracellular domain can be linked to a transmembranedomain. Other heterologous proteins of choice include, e.g., greenfluorescent protein, 0-gal, glutamate receptor, and the rhodopsinpresequence.

C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene or nucleic acid, suchas those cDNAs encoding a T2R family member, one typically subclones theT2R sequence into an expression vector that contains a strong promoterto direct transcription, a transcription/translation terminator, and iffor a nucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing the T2R protein areavailable in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.,Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kitsfor such expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available. In one embodiment,the eukaryotic expression vector is an adenoviral vector, anadeno-associated vector, or a retroviral vector.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is optionallypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the T2R-encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding a T2Rand signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. The nucleic acidsequence encoding a T2R may typically be linked to a cleavable signalpeptide sequence to promote secretion of the encoded protein by thetransformed cell. Such signal peptides would include, among others, thesignal peptides from tissue plasminogen activator, insulin, and neurongrowth factor, and juvenile hormone esterase of Heliothis virescens.Additional elements of the cassette may include enhancers and, ifgenomic DNA is used as the structural gene, introns with functionalsplice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early, promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as neomycin, hymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a sequence encoding a T2Rfamily member under the direction of the polyhedrin promoter or otherstrong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are optionally chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of a T2Rprotein, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressing aT2R gene.

In one preferred embodiment, a polynucleotide encoding a T2R is operablylinked to a EF-1α promoter, e.g., using a pEAK10 mammalian expressionvector (Edge Biosystems, MD) is used. Such vectors can be introducedinto cells, e.g., HEK-293 cells using any standard method, such astransfection using LipofectAMINE (Lifetechnologies).

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe T2R family member, which is recovered from the culture usingstandard techniques identified below.

IV. Purification of T2R Polypeptides

Either naturally occurring or recombinant T2R polypeptides can bepurified for use in functional assays. Optionally, recombinant T2Rpolypeptides are purified. Naturally occurring T2R polypeptides arepurified, e.g., from mammalian tissue such as tongue tissue, and anyother source of a T2R homolog. Recombinant T2R polypeptides are purifiedfrom any suitable bacterial or eukaryotic expression system, e.g., CHOcells or insect cells.

T2R proteins may be purified to substantial purity by standardtechniques, including selective precipitation with such substances asammonium sulfate; column chromatography, immunopurification methods, andothers (see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

A number of procedures can be employed when recombinant T2R familymembers are being purified. For example, proteins having establishedmolecular adhesion properties can be reversibly fused to the T2Rpolypeptide. With the appropriate ligand, a T2R can be selectivelyadsorbed to a purification column and then freed from the column in arelatively pure form. The fused protein is then removed by enzymaticactivity. Finally T2R proteins can be purified using immunoaffinitycolumns.

A. Purification of T2R Protein from Recombinant Cells

Recombinant proteins are expressed by transformed bacteria or eukaryoticcells such as CHO cells or insect cells in large amounts, typicallyafter promoter induction; but expression can be constitutive. Promoterinduction with IPTG is a one example of an inducible promoter system.Cells are grown according to standard procedures in the art. Fresh orfrozen cells are used for isolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of T2Rinclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. T2R polypeptides areseparated from other bacterial proteins by standard separationtechniques, e.g., with Ni-NTA agarose resin.

Alternatively, it is possible to purify T2R polypeptides from bacteriaperiplasm. After lysis of the bacteria, when a T2R protein is exportedinto the periplasm of the bacteria, the periplasmic fraction of thebacteria can be isolated by cold osmotic shock in addition to othermethods known to skill in the art. To isolate recombinant proteins fromthe periplasm, the bacterial cells are centrifuged to form a pellet. Thepellet is resuspended in a buffer containing 20% sucrose. To lyse thecells, the bacteria are centrifuged and the pellet is resuspended inice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10minutes. The cell suspension is centrifuged and the supernatant decantedand saved. The recombinant proteins present in the supernatant can beseparated from the host proteins by standard separation techniques wellknown to those of skill in the art.

B. Standard Protein Separation Techniques for Purifying T2R Polypeptides

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of a T2R protein can be used to isolate it fromproteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

T2R proteins can also be separated from other proteins on the basis ofits size, net surface charge; hydrophobicity, and affinity for ligands.In addition, antibodies raised against proteins can be conjugated tocolumn matrices and the proteins immunopurified. All of these methodsare well known in the art. It will be apparent to one of skill thatchromatographic techniques can be performed at any scale and usingequipment from many different manufacturers (e.g., Pharmacia Biotech).

V. Immunological Detection of T2R Polypeptides

In addition to the detection of T2R genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect T2R, e.g., to identify taste receptor cells, especially bittertaste receptor cells, and variants of T2R family members Immunoassayscan be used to qualitatively or quantitatively analyze the T2R. Ageneral overview of the applicable technology can be found in Harlow &Lane, Antibodies: A Laboratory Manual (1988).

A. Antibodies to T2R Family Members

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with a T2R family member are known to those of skill in theart (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow& Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice(2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

A number of T2R-comprising immunogens may be used to produce antibodiesspecifically reactive with a T2R family member. For example, arecombinant T2R protein, or an antigenic fragment thereof, is isolatedas described herein. Suitable antigenic regions include, e.g., theconserved motifs that are used to identify members of the T2R family,i.e., SEQ ID NOS:166, 167, 168, 169, 170, and 171. Recombinant proteincan be expressed in eukaryotic or prokaryotic cells as described above,and purified as generally described above. Recombinant protein is thepreferred immunogen for the production of monoclonal or polyclonalantibodies. Alternatively, a synthetic peptide derived from thesequences disclosed herein and conjugated to a carrier protein can beused an immunogen. Naturally occurring protein may also be used eitherin pure or impure form. The product is then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies may be generated, for subsequent use in immunoassays tomeasure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the T2R. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be done if desired (see Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-T2R proteins,or even other T2R family members or other related proteins from otherorganisms, using a competitive binding immunoassay. Specific polyclonalantisera and monoclonal antibodies will usually bind with a K_(d) of atleast about 0.1 mM, more usually at least about 1 μM, optionally atleast about 0.1 μM or better, and optionally 0.01 μM or better.

Once T2R family member specific antibodies are available, individual T2Rproteins can be detected by a variety of immunoassay methods. For areview of immunological and immunoassay procedures, see Basic andClinical Immunology (Stites & Terr eds., 7th ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio, ed., 1980); and Harlow & Lane, supra.

B. Immunological Binding Assays

T2R proteins can be detected and/or quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology: Antibodies inCell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7th ed. 1991). Immunological binding assays (orimmunoassays) typically use an antibody that specifically binds to aprotein or antigen of choice (in this case a T2R family member or anantigenic subsequence thereof). The antibody (e.g., anti-T2R) may beproduced by any of a number of means well known to those of skill in theart and as described above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled T2R polypeptide or alabeled anti-T2R antibody. Alternatively, the labeling agent may be athird moiety, such a secondary antibody, that specifically binds to theantibody/T2R complex (a secondary antibody is typically specific toantibodies of the species from which the first antibody is derived).Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins exhibit a strong non-immunogenic reactivity withimmunoglobulin constant regions from a variety of species (see, e.g.,Kronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J.Immunol. 135:2589-2542 (1985)). The labeling agent can be modified witha detectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, optionally from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-Competitive Assay Formats

Immunoassays for detecting a T2R protein in a sample may be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of antigen is directly measured. In one preferred“sandwich” assay, for example, the anti-T2R antibodies can be bounddirectly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture the T2R protein present in the testsample. The T2R protein is thus immobilized is then bound by a labelingagent, such as a second T2R antibody bearing a label. Alternatively, thesecond antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second or third antibody istypically modified with a detectable moiety, such as biotin, to whichanother molecule specifically binds, e.g., streptavidin, to provide adetectable moiety.

Competitive Assay Formats

In competitive assays, the amount of T2R protein present in the sampleis measured indirectly by measuring the amount of a known, added(exogenous) T2R protein displaced (competed away) from an anti-T2Rantibody by the unknown T2R protein present in a sample. In onecompetitive assay, a known amount of T2R protein is added to a sampleand the sample is then contacted with an antibody that specificallybinds to the T2R. The amount of exogenous T2R protein bound to theantibody is inversely proportional to the concentration of T2R proteinpresent in the sample. In a particularly preferred embodiment, theantibody is immobilized on a solid substrate. The amount of T2R proteinbound to the antibody may be determined either by measuring the amountof T2R protein present in a T2R/antibody complex, or alternatively bymeasuring the amount of remaining uncomplexed protein. The amount of T2Rprotein may be detected by providing a labeled T2R molecule:

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known T2R protein is immobilized on a solid substrate. Aknown amount of anti-T2R antibody is added to the sample, and the sampleis then contacted with the immobilized T2R. The amount of anti-T2Rantibody bound to the known immobilized T2R protein is inverselyproportional to the amount of T2R protein present in the sample. Again,the amount of immobilized antibody may be detected by detecting eitherthe immobilized fraction of antibody or the fraction of the antibodythat remains in solution. Detection may be direct where the antibody islabeled or indirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

Cross-Reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a protein at leastpartially encoded by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:57,SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82,SEQ ID NO:84, SEQ ID NO:86; SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92,SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102,SEQ ID NO:104 SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ IDNO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128,SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ IDNO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156,SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, or SEQ IDNO:165, can be immobilized to a solid support. Proteins (e.g., T2Rproteins and homologs) are added to the assay that compete for bindingof the antisera to the immobilized antigen. The ability of the addedproteins to compete for binding of the antisera to the immobilizedprotein is compared to the ability of the T2R polypeptide encoded by SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86; SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ IDNO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104 SEQ IDNO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:120, SEQ ID NO:122,SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ IDNO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150,SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:157, SEQ IDNO:159, SEQ ID NO:161, SEQ ID NO:163, or SEQ ID NO:165 to compete withitself. The percent crossreactivity for the above proteins iscalculated, using standard calculations. Those antisera with less than10% crossreactivity with each of the added proteins listed above areselected and pooled. The cross-reacting antibodies are optionallyremoved from the pooled antisera by immunoabsorption with the addedconsidered proteins, e.g., distantly related homologs. In addition,peptides comprising amino acid sequences representing conserved motifsthat are used to identify members of the T2R family can be used incross-reactivity determinations, i.e., SEQ ID NO:166, SEQ ID NO:167, SEQID NO:168; SEQ ID NO:169, SEQ ID NO:170, or SEQ ID NO:171.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of a T2R familymember, to the immunogen protein (i.e., T2R protein encoded by SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:34,SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45,SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:61, SEQ ID NO:63,SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86;SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96,SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104 SEQ ID NO:106,SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ IDNO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:120, SEQ ID NO:122, SEQID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132,SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ IDNO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:159,SEQ ID NO:161, SEQ ID NO:163, or SEQ ID NO:165). In order to make thiscomparison, the two proteins are each assayed at a wide range ofconcentrations and the amount of each protein required to inhibit 50% ofthe binding of the antisera to the immobilized protein is determined. Ifthe amount of the second protein required to inhibit 50% of binding isless than 10 times the amount of the protein encoded by SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86; SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104 SEQ ID NO:106, SEQ IDNO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQID NO:118, SEQ ID NO:120, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ IDNO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152,SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:159, SEQ IDNO:161, SEQ ID NO:163, or SEQ ID NO:165 that is required to inhibit 50%of binding, then the second protein is said to specifically bind to thepolyclonal antibodies generated to a T2R immunogen.

Antibodies raised against SEQ ID NOs:166-171 can also be used to prepareantibodies that specifically bind only to GPCRs of the T2R family, butnot to GPCRs from other families.

Polyclonal antibodies that specifically bind to a particular member ofthe T2R family, e.g., T2R01, can be make by subtracting outcross-reactive antibodies using other T2R family members.Species-specific polyclonal antibodies can be made in a similar way. Forexample, antibodies specific to human T2R01 can be made by subtractingout antibodies that are cross-reactive with orthologous sequences, e.g.,rat T2R01 or mouse T2R19.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of T2R protein in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind the T2R protein. The anti-T2R polypeptideantibodies specifically bind to the T2R polypeptide on the solidsupport. These antibodies may be directly labeled or alternatively maybe subsequently detected using labeled antibodies (e.g., labeled sheepanti-mouse antibodies) that specifically bind to the anti-T2Rantibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize a T2R protein,or secondary antibodies that recognize anti-T2R.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

VI. Assays for Modulators of T2R Family Members

A. Assays for T2R Protein Activity

T2R family members and their alleles and polymorphic variants areG-protein coupled receptors that participate in taste transduction,e.g., bitter taste transduction. The activity of T2R polypeptides can beassessed using a variety of in vitro and in vivo assays to determinefunctional, chemical, and physical effects, e.g., measuring ligandbinding (e.g., radioactive ligand binding), second messengers (e.g.,cAMP, cGMP, IP₃, DAG, or Ca²⁺), ion flux, phosphorylation levels,transcription levels, neurotransmitter levels, and the like.Furthermore, such assays can be used to test for inhibitors andactivators of T2R family members. Modulators can also be geneticallyaltered versions of T2R receptors. Such modulators of taste transductionactivity are useful for customizing taste, for example to modify thedetection of bitter tastes.

The T2R protein of the assay will typically be selected from apolypeptide having a sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5;SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:160, SEQ IDNO:162, or SEQ ID NO:164 or conservatively modified variant thereof.

Alternatively, the T2R protein of the assay will be derived from aeukaryote and include an amino acid subsequence having amino acidsequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7,SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:160, SEQ IDNO:162, or SEQ ID NO:164. Generally, the amino acid sequence identitywill be at least 60%, optionally at least 70% to 85%, optionally atleast 90-95%. Optionally, the polypeptide of the assays will comprise adomain of a T2R protein, such as an extracellular domain, transmembraneregion, transmembrane domain, cytoplasmic domain, ligand binding domain,subunit association domain, active site, and the like. Either the T2Rprotein or a domain thereof can be covalently linked to a heterologousprotein to create a chimeric protein used in the assays describedherein.

Modulators of T2R receptor activity are tested using T2R polypeptides asdescribed above, either recombinant or naturally occurring. The proteincan be isolated, expressed in a cell, expressed in a membrane derivedfrom a cell, expressed in tissue or in an animal, either recombinant ornaturally occurring. For example, tongue slices, dissociated bells froma tongue, transformed cells, or membranes can b used. Modulation istested using one of the in vitro or in vivo assays described herein.Taste transduction can also be examined in vitro with soluble or solidstate reactions; using a full-length T2R-GPCR or a chimeric moleculesuch as an extracellular domain or transmembrane region, or combinationthereof, of a T2R receptor covalently linked to a heterologous signaltransduction domain, or a heterologous extracellular domain and/ortransmembrane region covalently linked to the transmembrane and/orcytoplasmic domain of a T2R receptor. Furthermore, ligand-bindingdomains of the protein of interest can be used in vitro in soluble orsolid state reactions to assay for ligand binding. In numerousembodiments, a chimeric receptor will be made that comprises all or partof a T2R polypeptide as well an additional sequence that facilitates thelocalization of the T2R to the membrane, such as a rhodopsin, e.g., anN-terminal fragment of a rhodopsin protein.

Ligand binding to a T2R protein, a domain, or chimeric protein can betested in solution, in a bilayer membrane, attached to a solid phase, ina lipid monolayer, or in vesicles. Binding of a modulator can be testedusing, e.g., changes in spectroscopic characteristics (e.g.,fluorescence, absorbance, refractive index) hydrodynamic (e.g., shape),chromatographic, or solubility properties.

Receptor-G-protein interactions can also be examined. For example,binding of the G-protein to the receptor or its release from thereceptor can be examined. For example, in the absence of GTP, anactivator will lead to the formation of a tight complex of a G protein(all three subunits) with the receptor. This complex can be detected ina variety of ways, as noted above. Such an assay can be modified tosearch for inhibitors, e.g., by adding an activator to the receptor andG protein in the absence of GTP, which form a tight complex, and thenscreen for inhibitors by looking at dissociation of the receptor-Gprotein complex. In the presence of GTP, release of the alpha subunit ofthe G protein from the other two G protein subunits serves as acriterion of activation.

In particularly preferred embodiments, T2R-Gustducin interactions aremonitored as a function of T2R receptor activation. As shown in ExampleIX, mouse T2R5 shows strong cycloheximide-dependent coupling withGustducin. Such ligand dependent coupling of T2R receptors withGustducin can be used as a marker to identify modifiers of any member ofthe T2R family.

An activated or inhibited G-protein will in turn alter the properties oftarget enzymes, channels, and other effector proteins. The classicexamples are the activation of cGMP phosphodiesterase by transducin inthe visual system, adenylate cyclase by the stimulatory G-protein,phospholipase C by Gq and other cognate G proteins, and modulation ofdiverse channels by Gi and other G proteins. Downstream consequences canalso be examined such as generation of diacyl glycerol and IP3 byphospholipase C, and in turn, for calcium mobilization by IP3.

In a preferred embodiment, a T2R polypeptide is expressed in aeukaryotic cell as a chimeric receptor with a heterologous, chaperonesequence that facilitates its maturation and targeting through thesecretory pathway. In a preferred embodiment, the heterologous sequenceis a rhodopsin sequence, such as an N-terminal fragment of a rhodopsin.Such chimeric T2R receptors can be expressed in any eukaryotic cell,such as HEK-293 cells. Preferably, the cells comprise a functional Gprotein, e.g., Gα15, that is capable of coupling the chimeric receptorto an intracellular signaling pathway or to a signaling protein such asphospholipase Cβ. Activation of such chimeric receptors in such cellscan be detected using any standard method, such as by detecting changesin intracellular calcium by detecting FURA-2 dependent fluorescence inthe cell.

Activated GPCR receptors become substrates for kinases thatphosphorylate the C-terminal tail of the receptor. (and possibly othersites as well). Thus, activators will promote the transfer of ³²P fromgamma-labeled GTP to the receptor, which can be assayed with ascintillation counter. The phosphorylation of the C-terminal tail willpromote the binding of arrestin-like proteins and will interfere withthe binding of G-proteins. The kinase/arrestin pathway plays a key rolein the desensitization of many GPCR receptors. For example, compoundsthat modulate the duration a taste receptor stays active would be usefulas a means of prolonging a desired taste or cutting off an unpleasantone. For a general review of GPCR signal transduction and methods ofassaying signal transduction, see, e.g., Methods in Enzymology, vols.237 and 238 (1994) and volume 96 (1983); Bourne et al., Nature10:349:117-27 (1991); Bourne et al., Nature 348:125-32 (1990); Pitcheret al., Annu. Rev. Biochem. 67:653-92 (1998).

Samples or assays that are treated with a potential T2R proteininhibitor or activator are compared to control samples without the testcompound, to examine the extent of modulation. Such assays may becarried out in the presence of a bitter tastant that is known toactivate the particular receptor, and modulation of thebitter-tastant-dependent activation monitored. Control samples(untreated with activators or inhibitors) are assigned a relative T2Ractivity value of 100. Inhibition of a T2R protein is achieved when theT2R activity value relative to the control is about 90%, optionally 50%,optionally 25-0%. Activation of a T2R protein is achieved when the T2Ractivity value relative to the control is 110%, optionally 150%,200-500%, or 1000-2000%.

Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing a T2R protein. One means to determine changes in cellularpolarization is by measuring changes in current (thereby measuringchanges in polarization) with voltage-clamp and patch-clamp techniques,e.g., the “cell-attached” mode, the “inside-out” mode, and the “wholecell” mode (see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595(1997)). Whole cell currents are conveniently determined using thestandard methodology (see, e.g., Hamil et al., PFlugers. Archiv. 391:85(1981). Other known assays include: radiolabeled ion flux assays andfluorescence assays using voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Gonzales &Tsien, Chem. Biol. 4:269-277 (1997); Daniel et al., J. Pharmacol. Meth.25:185-193 (1991); Holevinsky et al., J. Membrane Biology 137:59-70(1994)). Generally, the compounds to be tested are present in the rangefrom 1 pM to 100 mM.

The effects of the test compounds upon the function of the polypeptidescan be measured by examining any of the parameters described above. Anysuitable physiological change that affects GPCR activity can be used toassess the influence of a test compound on the polypeptides of thisinvention. When the functional consequences are determined using intactcells or animals, one can also measure a variety of effects such astransmitter release, hormone release, transcriptional changes to bothknown and uncharacterized genetic markers (e.g., northern blots),changes in cell metabolism such as cell growth or pH changes, andchanges in intracellular second messengers such as Ca²⁺, IP3, cGMP, orcAMP.

Preferred assays for G-protein coupled receptors include cells that areloaded with ion or voltage sensitive dyes to report receptor activity.Assays for determining activity of such receptors can also use knownagonists and antagonists for other G-protein coupled receptors asnegative or positive controls to assess activity of tested compounds. Inassays for identifying modulatory compounds (e.g., agonists,antagonists), changes in the level of ions in the cytoplasm or membranevoltage will be monitored using an ion sensitive or membrane voltagefluorescent indicator, respectively. Among the ion-sensitive indicatorsand voltage probes that may be employed are those disclosed in theMolecular Probes 1997 Catalog. For G-protein coupled receptors,promiscuous G-proteins such as Gα15 and Gα16 can be used in the assay ofchoice (Wilkie et al., Proc. Nat'l Acad. Sci. USA 88:10049-10053(1991)). Such promiscuous G-proteins allow coupling of a wide range ofreceptors.

Receptor activation typically initiates subsequent intracellular events,e.g., increases in second messengers such as IP3, which releasesintracellular stores of calcium ions. Activation of some G-proteincoupled receptors stimulates the formation of inositol triphosphate(IP3) through phospholipase C-mediated hydrolysis ofphosphatidylinositol (Berridge & Irvine, Nature 312:315-21 (1984)). IP3in turn stimulates the release of intracellular calcium ion stores.Thus, a change in cytoplasmic calcium ion levels, or a change in secondmessenger levels such as IP3 can be used to assess G-protein coupledreceptor function. Cells expressing such G-protein coupled receptors mayexhibit increased cytoplasmic calcium levels as a result of contributionfrom both intracellular stores and via activation of ion channels, inwhich case it may be desirable although not necessary to conduct suchassays in calcium-free buffer, optionally supplemented with a chelatingagent such as EGTA, to distinguish fluorescence response resulting fromcalcium release from internal stores.

Other assays can involve determining the activity of receptors which,when activated, result in a change in the level of intracellular cyclicnucleotides, e.g., cAMP or cGMP, by activating or inhibiting enzymessuch as adenylate cyclase. There are cyclic nucleotide-gated ionchannels, e.g., rod photoreceptor cell channels and olfactory neuronchannels that are permeable to cations upon activation by binding ofcAMP or cGMP (see, e.g., Altenhofen et al., Proc. Natl. Acad. Sci.U.S.A. 88:9868-9872 (1991) and Dhallan et al., Nature 347:184-187(1990)). In cases where activation of the receptor results in a decreasein cyclic nucleotide levels, it may be preferable to expose the cells toagents that increase intracellular cyclic nucleotide levels, e.g.,forskolin, prior to adding a receptor-activating compound to the cellsin the assay. Cells for this type of assay can be made byco-transfection of a host cell with DNA encoding a cyclicnucleotide-crated ion channel, GPCR phosphatase and DNA encoding areceptor (e.g., certain glutamate receptors, muscarinic acetylcholinereceptors, dopamine receptors, serotonin receptors, and the like),which, when activated, causes a change in cyclic nucleotide levels inthe cytoplasm.

In a preferred embodiment, T2R protein activity is measured byexpressing a T2R gene in a heterologous cell with a promiscuousG-protein that links the receptor to a phospholipase C signaltransduction pathway (see Offermanns & Simon, J. Biol. Chem.270:15175-15180 (1995)). Optionally the cell line is HEK-293 (which doesnot naturally express T2R genes) and the promiscuous G-protein is Gα15(Offermanns & Simon, supra). Modulation of taste transduction is assayedby measuring changes in intracellular Ca²⁺ levels, which change inresponse to modulation of the T2R signal transduction pathway viaadministration of a molecule that associates with a T2R protein. Changesin Ca²⁺ levels are optionally measured using fluorescent Ca²⁺ indicatordyes and fluorometric imaging.

In one embodiment, the changes in intracellular cAMP or cGMP can bemeasured using immunoassays. The method described in Offermanns & Simon,J. Biol. Chem. 270:15175-15180 (1995) may be used to determine the levelof cAMP. Also, the method described in Felley-Bosco et al., Am. J. Resp.Cell and Mol. Biol. 11:159-164 (1994) may be used to determine the levelof cGMP. Further, an assay kit for measuring cAMP and/or cGMP isdescribed in U.S. Pat. No. 4,115,538, herein incorporated by reference.

In another embodiment, phosphatidyl inositol (PI) hydrolysis can beanalyzed according to U.S. Pat. No. 5,436,128, herein incorporated byreference. Briefly, the assay involves labeling of cells with³H-myoinositol for 48 or more hrs. The labeled cells are treated with atest compound for one hour. The treated cells are lysed and extracted inchloroform-methanol-water after which the inositol phosphates wereseparated by ion exchange chromatography and quantified by scintillationcounting. Fold stimulation is determined by calculating the ratio of cpmin the presence of agonist to cpm in the presence of buffer control.Likewise, fold inhibition is determined by calculating the ratio of cpmin the presence of antagonist to cpm in the presence of buffer control(which may or may not contain an agonist).

In another embodiment, transcription levels can be measured to assessthe effects of a test compound on signal transduction. A host cellcontaining a T2R protein of interest is contacted with a test compoundfor a sufficient time to effect any interactions, and then the level ofgene expression is measured. The amount of time to effect suchinteractions may be empirically determined, such as by running a timecourse and measuring the level of transcription as a function of time.The amount of transcription may be measured by using any method known tothose of skill in the art to be suitable.

For example, mRNA expression of the protein of interest may be detectedusing northern blots or their polypeptide products may be identifiedusing immunoassays. Alternatively, transcription based assays usingreporter gene may be used as described in U.S. Pat. No. 5,436,128,herein incorporated by reference. The reporter genes can be, e.g.,chloramphenicol acetyltransferase, luciferase, β-galactosidase andalkaline phosphatase. Furthermore, the protein of interest can be usedas an indirect reporter via attachment to a second reporter such asgreen fluorescent protein (see, e.g., Mistili & Spector, NatureBiotechnology 15:961-964 (1997)).

The amount of transcription is then compared to the amount oftranscription in either the same cell in the absence of the testcompound, or it may be compared with the amount of transcription in asubstantially identical cell that lacks the protein of interest. Asubstantially identical cell may be derived from the same cells fromwhich the recombinant cell was prepared but which had not been modifiedby introduction of heterologous DNA. Any difference in the amount oftranscription indicates that the test compound has in some Dimmeraltered the activity of the protein of interest.

B. Modulators

The compounds tested as modulators of a T2R family member can be anysmall chemical compound, or a biological entity, such as a protein,sugar, nucleic acid or lipid. Alternatively, modulators can begenetically altered versions of a T2R gene. Typically, test compoundswill be small chemical molecules and peptides. Essentially any chemicalcompound can be used as a potential modulator or ligand in the assays ofthe invention, although most often compounds can be dissolved in aqueousor organic (especially DMSO-based) solutions are used. The assays aredesigned to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source to assays,which are typically run in parallel (e.g., in microtiter formats onmicrotiter plates in robotic assays). It will be appreciated that thereare many suppliers of chemical compounds, including Sigma (St. Louis,Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), FlukaChemika-Biochemica Analytika (Buchs, Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan.18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

C. Solid State and Soluble High Throughput Assays

In one embodiment the invention provide soluble assays using moleculessuch as a domain such as ligand binding domain, an extracellular domain,a transmembrane domain (e.g., one comprising seven transmembrane regionsand cytosolic loops), the transmembrane domain and a cytoplasmic domain,an active site, a subunit association region, etc.; a domain that iscovalently linked to a heterologous protein to create a chimericmolecule; a T2R protein; or a cell or tissue expressing a T2R protein,either naturally occurring or recombinant. In another embodiment, theinvention provides solid phase based in vitro assays in a highthroughput format, where the domain, chimeric molecule, T2R protein, orcell or tissue expressing the T2R is attached to a solid phasesubstrate.

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100-about 1500different compounds. It is possible to assay several different platesper day; assay screens for up to about 6,000-20,000 different compoundsis possible using the integrated systems of the invention. Morerecently, microfluidic approaches to reagent manipulation have beendeveloped.

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non covalent linkage, e.g., viaa tag. The tag can be any of a variety of components. In general, amolecule which binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest (e.g., the taste transductionmolecule of interest) is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethelyne glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

D. Computer-Based Assays

Yet another assay for compounds that modulate T2R protein activityinvolves computer assisted drug design, in which a computer system isused to generate a three-dimensional structure of a T2R protein based onthe structural information encoded by its amino acid sequence. The inputamino acid sequence interacts directly and actively with apreestablished algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind, e.g., ligands. These regionsare then used to identify ligands that bind to the protein.

The three-dimensional structural model of the protein is generated byentering protein amino acid sequences of at least 10 amino acid residuesor corresponding nucleic acid sequences encoding a T2R polypeptide intothe computer system. The nucleotide sequence encoding the polypeptide,or the amino acid sequence thereof, can be any of SEQ ID NO:1-165, andconservatively modified versions thereof. The amino acid sequencerepresents the primary sequence or subsequence of the protein, whichencodes the structural information of the protein. At least 10 residuesof the amino acid sequence (or a nucleotide sequence encoding 10 aminoacids) are entered into the computer system from computer keyboards,computer readable substrates that include, but are not limited to,electronic storage media (e.g., magnetic diskettes, tapes, cartridges,and chips), optical media (e.g., CD ROM), information distributed byinternet sites, and by RAM. The three-dimensional structural model ofthe protein is then generated by the interaction of the amino acidsequence and the computer system, using software known to those of skillin the art.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the protein of interest. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potentials, hydrophobic potentials, solventaccessible surfaces, and hydrogen bonding. Secondary energy termsinclude van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the baths of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino-acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of the T2R protein to identify ligands that bind to the protein.Binding affinity between the protein and ligands is determined usingenergy terms to determine which ligands have an enhanced probability ofbinding to the protein.

Computer systems are also used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of T2R genes. Such mutationscan be associated with disease states or genetic traits. As describedabove, GeneChip™ and related technology can also be used to screen formutations, polymorphic variants, alleles and interspecies homologs. Oncethe variants are identified, diagnostic assays can be used to identifypatients having such mutated genes. Identification of the mutated T2Rgenes involves receiving input of a first nucleic acid or amino acidsequence of a T2R gene, e.g., any of SEQ ID NO:1-165, or conservativelymodified versions thereof. The sequence is entered into the computersystem as described above. The first nucleic acid or amino acid sequenceis then compared to a second nucleic acid or amino acid sequence thathas substantial identity to the first sequence. The second sequence isentered into the computer system in the manner described above. Once thefirst and second sequences are compared, nucleotide or amino aciddifferences between the sequences are identified. Such sequences canrepresent allelic differences in various T2R genes, and mutationsassociated with disease states and genetic traits.

IX. Administration and Pharmaceutical Compositions

Taste modulators can be administered directly to the mammalian subjectfor modulation of taste, e.g., modulation of bitter taste, in vivo.Administration is by any of the routes normally used for introducing amodulator compound into ultimate contact with the tissue to be treated,optionally the tongue or mouth. The taste modulators are administered inany suitable manner, optionally with pharmaceutically acceptablecarriers. Suitable methods of administering such modulators areavailable and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed. 1985)).

The taste modulators, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, by orally, topically, intravenously,intraperitoneally, intravesically or intrathecally. Optionally, thecompositions are administered orally or nasally. The formulations ofcompounds can be presented in unit-dose or multi-dose sealed containers,such as ampules and vials. Solutions and suspensions can be preparedfrom sterile powders, granules, and tablets of the kind previouslydescribed. The modulators can also be administered as part a of preparedfood or drug.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial response in thesubject over time. The dose will be determined by the efficacy of theparticular taste modulators employed and the condition of the subject,as well as the body weight or surface area of the area to be treated.The size of the dose also will be determined by the existence, nature,and extent of any adverse side-effects that accompany the administrationof a particular compound or vector in a particular subject.

In determining the effective amount of the modulator to be administeredin a physician may evaluate circulating plasma levels of the modulator,modulator toxicities, and the production of anti-modulator antibodies.In general, the dose equivalent of a modulator is from about 1 ng/kg to10 mg/kg for a typical subject.

For administration, taste modulators of the present invention can beadministered at a rate determined by the LD-50 of the modulator, and theside-effects of the inhibitor at various concentrations, as applied tothe mass and overall health of the subject. Administration can beaccomplished via single or divided doses.

VIII. Kits

T2R genes and their homologs are useful tools for identifying tastereceptor cells, for forensics and paternity determinations, and forexamining taste transduction. T2R family member-specific reagents thatspecifically hybridize to T2R nucleic acids, such as T2R probes andprimers, and T2R specific reagents that specifically bind to a T2Rprotein, e.g., T2R antibodies are used to examine taste cell expressionand taste transduction regulation.

Nucleic acid assays for the presence of DNA and RNA for a T2R familymember in a sample include numerous techniques are known to thoseskilled in the art, such as Southern analysis, northern analysis, dotblots, RNase protection, S1 analysis, amplification techniques such asPCR and LCR, and in situ hybridization. In in situ hybridization, forexample, the target nucleic acid is liberated from its cellularsurroundings in such as to be available for hybridization within thecell while preserving the cellular morphology for subsequentinterpretation and analysis. The following articles provide an overviewof the art of in situ hybridization: Singer et al., Biotechniques4:230-250 (1986); Haase et al., Methods in Virology, vol. VII, pp.189-226 (1984); and Nucleic Acid Hybridization: A Practical Approach(Hames et al., eds. 1987). In addition, a T2R protein can be detectedwith the various immunoassay techniques described above. The test sampleis typically compared to both a positive control (e.g., a sampleexpressing a recombinant T2R protein) and a negative control.

The present invention also provides for kits for screening formodulators of T2R family members. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: T2R nucleic acids orproteins, reaction tubes, and instructions for testing T2R activity.Optionally, the kit contains a biologically active T2R receptor. A widevariety of kits and components can be prepared according to the presentinvention, depending upon the intended user of the kit and theparticular needs of the user.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example I Identification of the T2R Gene Family

Recent genetic linkage studies in humans identified a locus at 5p15 thatis associated with the ability to respond to the bitter substance6-n-propyl-2-thiouracil (PROP; Reed et al., Am. J. Hum. Genet.64:1478-1480 (1999)). To determine whether differences in PROPsensitivity reflected functional differences in a bitter taste receptor,DNA sequence databases were searched for genes encoding candidatetransmembrane proteins at this location. Analysis of open reading framesin 450 kb of DNA spanning six sequenced human genomic BAC clones (see,e.g., accession number AC003015) from this interval identified a novelGPCR (T2R1) at 5p15.2. T2R1 has seven putative transmembrane segments aswell as several conserved residues often present in GPCRs (Probst etal., DNA Cell. Biol. 11:1-20 (1992)).

Computer searches using T2R1, and reiterated with T2R1-relatedsequences, revealed 19 additional human receptors (12 full-length and 7pseudogenes). Full-length hT2Rs were isolated by PCR amplification ofgenomic DNA. Full-length hT2Rs were used to probe a rat circumvallatecDNA library (Hoon et al., Cell, 96:541-551 (1999)) and mouse BAC filterarrays (Genome Systems) at low stringency (50-55° C. wash in 1×SSC).Southern hybridization experiments were used to identify a non-redundantset of positive BACs and to order overlapping BACs.

These new receptors, referred to as T2Rs (also known as “SF”), define anovel family of GPCRs that are distantly related to V1R vomeronasalreceptors and opsins. In contrast to T1Rs, which belong to thesuperfamily of GPCRs characterized by a large N-terminal domain (Hoon etal., Cell, 96:541-551 (1999)), the T2Rs have only a short extracellularN-terminus. Individual members of the T2R-family exhibit 30-70% aminoacid identity, and most share highly conserved sequence motifs in thefirst three and last transmembrane segments, and also in the secondcytoplasmic loop. The most divergent regions between T2Rs are theextracellular segments, extending partway into the transmembranehelices. Presumably, the high degree of variability between T2Rsreflects the need to recognize many structurally diverse ligands. Likemany other GPCR genes, T2Rs do not contain introns that interrupt codingregions.

Example II Organization of Human T2R Genes

The identified human T2R genes are localized on three chromosomes, andare often organized as head-to-tail arrays. For example, four receptorgenes are clustered within a single PAC clone from 7q31 and nine in aBAC clone from 12p13. There may be more human T2Rs in these arrays, asseveral additional human T2Rs were found within partially sequenced BACclones that overlap the 9 gene T2R cluster. Within a given array, thesimilarity of receptors is highly variable, including both relativelyrelated (e.g. hT2R₁₃, hT2R₁₄ and hT2R₁₅), and highly divergent receptors(e.g. hT2R₃ and hT2R₄). This type of organization is mirrored in themouse (see below), and resembles the genomic organization that has beenobserved for olfactory receptor genes in humans, mice, flies and worms(Rouquier et al., Nat. Genet. 18:243-250 (1998)); Sullivan et al., PNAS93:884-888 (1996)); Clyne et al., Neuron 22:327-388 (1999)); Vosshall etal., Cell 96:725-736 (1999)); Troemel et al., Cell 83:207-218 (1995)).

To obtain estimates of the size of this gene family, various genomicresources were examined. Analysis of the Genome Sequence Survey database(gss) yielded 12 partial T2R sequences. Because this database representsan essentially random sampling of ˜14% of the human genome, this numbersuggests tha there may be ˜90 T2R genes in the human genome. Similarsearches of the finished (nr) and unfinished high-throughput humangenomic sequence databases (htgs) produced 36 full-length and 15 partialT2R sequences. These databases contain ˜50% of the genome sequence, alsopointing to ˜100 T2R genes in the genome. Recognizing that this analysismay be inaccurate due to the quality of the available databases, and theclustered, non-random distribution of T2Rs in the human genome, it isestimated that the T2R family consists of between 80 to 120 members.However, more than ⅓ of the full-length human T2Rs are pseudogenes;thus, the final number of functional human receptors: may besignificantly smaller (i.e., 40-80). This is similar to what has beenobserved for human olfactory receptors, where many of the genes appearto be pseudogenes (Rouquier et al., Nat. Genet. 18:243-250 (1998)).

Example III T2R Genes are Linked to Loci Involved in Bitter Taste

The genetics of sweet and bitter tasting has been extensively studied inmice, where a number of loci influencing responses to sweet and bittertastants have been mapped by behavioral taste-choice assays (Warren andLewis, Nature 227:77-78 (1970)); Fuller, J. Hered. 65:33-66 (1974)). Thedistal end of mouse chromosome 6 contains a cluster of bitter genes thatincludes Soa (for sucrose octaacetate; Capeless et al., Behav. Genet.22:655-663 (1992)), Rua (raffinose undecaacetate; Lush, Genet. Res.47:117-123 (1986)), Cyx (cycloheximide; Lush and Holland, Genet. Res.52:207-212(1988)) and Qui (quinine; Lush, Genet. Res. 44:151-160(1984)). Recombination studies indicated that these four loci areclosely linked to each other, and to Prp (salivary proline rich protein;Azen et al., Trends Genet. 2:199-200 (1986)). The human 9 gene T2Rcluster contains three interspersed PRP genes, and maps to an intervalthat is homologous with the mouse chromosome 6 bitter cluster.

To define the relationship between the mouse chromosome 6 bitter clusterand T2Rs, a large number of mouse T2R genes were isolated and theirgenomic organization and physical and genetic map locations weredetermined. By screening mouse genomic libraries with human T2Rs, 61BAC-clones containing 28 mouse T2Rs were isolated. The mouse and humanreceptors display significant amino acid sequence divergence, but sharethe sequence motifs common to members of this novel family of receptors.Mouse T2Rs were mapped using a mouse/hamster radiation hybrid panel(Research Genetics), and by examining the strain distribution pattern ofsingle nucleotide polymorphisms in a panel of C57BL/6J×DBA/2Jrecombinant inbred lines (Jackson Laboratory). These studies showed thatthe mouse genes are clustered at only a few genomic locations. Eachgenomic interval containing mouse T2Rs is homologous to one containingits closest human counterpart: mT2R₈ and hT2R₄, mT2R₁₈ and hT2R₁₆, andmT2R₁₉ and hT2R₁. Of these 3 sets of potentially orthologous pairs ofhuman/mouse receptors, both the human T2R₁ and T2R₁₆ genes map tolocations implicated in human bitter perception (Conneally et al., Hum.Hered. 26:267-271 (1976); Reed et al., Am. J. Hum. Genet. 64:1478-1480(1999)). The remaining 25 mT2Rs all map to the distal end of chromosome6, and are represented by 3 BAC contigs spanning at least 400 kb.

Since Prp and the bitter-cluster also map to the distal end of mousechromosome 6, it was determined whether they localize within this arrayof T2Rs. Analysis of a DBA/2×C57BL/6 recombinant inbred panel revealedthat receptors within all 3 BAC-contigs co-segregate with Prp and thebitter cluster. Further, the mouse Prp gene was isolated (accessionnumber M23236, containing D6Mit13) and shown that it lies within thelarge chromosome 6 T2R cluster. These results demonstrate that T2Rs areintimately linked to loci implicated in bitter perception.

Example IV T2Rs are Expressed in Taste Receptor Cells

The lingual epithelium contains taste buds in three types of papillae:circumvallate papillae at the very back of the tongue, foliate papillaeat the posterior lateral edge of the tongue, and fungiform papillaedispersed throughout the front half of the tongue surface. Other partsof the oral cavity also have taste buds; these are particularlyprominent in the palate epithelium in an area known as thegeschmackstreifen and in the epiglottis. To examine the patterns ofexpression of T2Rs, in situ hybridizations were performed using sectionsof various taste papillae. To ensure that the probes used were expressedin taste tissue, a rat circumvallate cDNA library was screened, leadingto the isolation of 14 rat T2Rs cDNAs, each of which is an ortholog of amouse genomic clone.

To carry out the in situ hybridization, tissue was obtained from adultrats and mice. No sex-specific differences of expression patterns wereobserved, therefore male and female animals were used interchangeably.Fresh frozen sections (16 μm) were attached to silanized slides andprepared for in situ hybridization as described previously (Hoon et al.,Cell, 96:541-551 (1999)). All in situ hybridizations were carried out athigh stringency (hybridization, 5×SSC, 50% formamide, 65-72° C.;washing, 0.2×SSC, 72° C.). Signals were developed usingalkaline-phosphatase conjugated antibodies to digoxigenin and standardchromogenic substrates (Boehringer Mannheim). Where possible, probescontained extensive 3′-non translated sequence to minimize potentialcross-hybridization between T2Rs, which was not observed at thestringency used for in situ hybridization.

These experiments demonstrated that T2Rs are selectively expressed insubsets of taste receptor cells of the tongue and palate epithelium.Each receptor hybridizes to an average of 2 cells per taste bud persection. Since the sections used in these experiments contain ⅕-⅓ thedepth of a taste bud, this reflects a total of 6-10 positive cells/tastebud/probe (or about 15% of the cells in a taste bud). Examination ofserial sections demonstrated that all of the taste buds of thecircumvallate papilla contain cells that are positive for each of theseprobes. Thus far, comparable results have been observed with 11 ratT2Rs, and in mouse sections hybridized with 17 different mT2R probes.

Similar studies in foliate, geschmackstreifen and epiglottis taste budsdemonstrated that each receptor probe also labels approximately 15% ofthe cells in every taste bud. In contrast, T2Rs are rarely expressed infungiform papillae. Examination of hundreds of fungiform taste budsusing 11 different T2R probes demonstrated that less than 10% of allfungiform papillae contain T2R-expressing cells. Interestingly, the fewfungiform taste buds that do express T2Rs regularly contain multiplepositive cells. In fact, the number of positive cells in these papillaeis not significantly different from that seen in taste buds from otherregions of the oral cavity. Furthermore; fungiform papillae that containT2R-expressing cells generally appear clustered. This unexpected findingmay provide an important clue about the logic of taste coding. It isknown that single fibers of the chorda tympani nerve innervate multiplecells in a fungiform taste bud, and that the same fiber often projectsto neighboring papillae (Miller, J. Comp. Neurol. 158:155-166 (1974)).Perhaps the non-random distribution of T2R-positive taste receptor cellsand taste buds in fungiform papillae reflect a map of connectivitybetween similar cells.

Northern analysis and in situ hybridization demonstrated that T2Rs arenot widely expressed outside taste tissue.

Example V Individual Receptor Cells Express Multiple T2R Receptors

The above-described results demonstrated that any given T2R is expressedin ˜15% of the cells of circumvallate, foliate and palate taste buds.Given that there are over 30 T2Rs in the rodent genome, a taste cellmust express more than one receptor. To determine how many receptors areexpressed in any cell, and what fraction of taste receptor cells expressT2Rs, the number of circumvallate cells labeled with various mixes of 2,5 or 10 receptors was compared with those labeled with the correspondingindividual probes. By counting positive cells in multiple serialsections, it was determined that the number of taste cells labeled withthe mixed probes (˜20%) was only slightly larger than that labeled byany individual receptor (˜15%). Not surprisingly, the signal intensitywas significantly enhanced in the mixed probe hybridizations. Similarresults were observed in taste buds from other regions of the oralcavity including the fungiform papillae. To directly demonstrateco-expression, double labeling experiments were carried out using acollection of differentially labeled cRNA probes. For double-labelfluorescent detection, probes were labeled either with fluorescein orwith digoxigenin. An alkaline-phosphatase conjugated anti-fluoresceinantibody (Amersham) and a horseradish-peroxidase conjugatedanti-digoxigenin antibody were used in combination with fast-red andtyramide fluorogenic substrates (Boehringer Mannheim and New EnglandNuclear). In these experiments, the majority of cells were found toexpress multiple receptors.

Example VI T2R Genes are Selectively Expressed in Gustducin-ExpressingCells

Previous results had shown that T1Rs are expressed in ˜30% of tastereceptor cells. In situ hybridizations with differentially labeled T1Rand T2R probes showed that there is no overlap in the expression ofthese two classes of receptors. Gustducin is also expressed in a largesubset of taste receptor cells, but for the most part is notco-expressed with T1Rs (Hoon et al., Cell, 96:541-551 (1999)). Todetermine if T2Rs are expressed in gustducin cells, in situhybridizations were performed using differentially labeled T2Rs andgustducin riboprobes. These experiments demonstrated that T2Rs areexclusively expressed in gustducin-positive cells of the tongue andpalate taste buds.

Approximately ⅓ of the gustducin cells in the circumvallate, foliate andpalate taste buds did not label with a mix of 10 T2R probes, suggestingthat not all gustducin-expressing cells express T2Rs. These cells mayexpress other, perhaps more distantly related receptors, or could be ata different developmental stage. In fungiform taste buds the situationis quite different. Since only 10% of fungiform taste buds contain T2Rpositive cells, the great majority of gustducin-positive cells in thefront of the tongue do not appear to co-express members of the T2Rfamily of receptors. Therefore, there is likely to be an additional setof receptors expressed in the gustducin-positive cells of fungiformpapillae.

Example VII Functional Expression of T2Rs

T2Rs were expressed in conjunction with Gα15, a G-protein α-subunit thathas been shown to couple a wide range of receptors to phospholipase Cβ(Offermanns and Simon, J Biol Chem, 270:15175-80 (1995); Krautwurst etal., Cell 95:917-926 (1998)). In this system, receptor activation leadsto increases in intracellular calcium [Ca2+]i, which can be monitored atthe single cell level using the FURA-2 calcium-indicator dye (Tsien etal., Cell Calcium 6:145-157 (1985)). To test and optimize Gα15 coupling,two different GPCRs, a Gαi-coupled μ-opioid receptor (Reisine,Neuropharm. 34:463-472 (1995)) and a Gαq-coupled mGluR1 receptor (Masuet al, Nature 349:760-765 (1991)), were used. Transfection of thesereceptors into HEK-293 cell produced robust, agonist-selective, andGα15-dependent Ca²⁺ responses (FIG. 1).

A number of studies have shown that many GPCRs, in particular sensoryreceptors, require specific “chaperones” for maturation and targetingthrough the secretory pathway (Baker et al., Embo J 13:4886-4895 (1994);Dwyer et al., Cell 93:455-466 (1998)). Recently, Krautwurst et al. (Cell95:917-926 (1998)) generated chimeric receptors consisting of the first20 amino acids of rhodopsin and various rodent olfactory receptors.These were targeted to the plasma membrane and functioned as odorantreceptors in HEK-293 cells. To determine whether rhodopsin sequences canalso help target T2Rs to the plasma membra, rhodopsin-T2R chimeras(rho-T2Rs) were constructed. Expression of these fusion proteinsdemonstrated that the first 39 amino acids of bovine rhodopsin are veryeffective in targeting T2Rs to the plasma membrane of HEK-293 cells(FIG. 2). Similar results were obtained with 11 human and 16 rodent T2Rs(see below). To further enhance the level of T2R expression, rho-T2Rswere placed under the control of a strong EF-1α promoter, and introducedas episomal plasmids into modified HEK-293 cells expressing Gα15(pEAKrapid cells).

A bridge overlap PCR extension technique was used to generate rho-T2Rchimeras, which contain the first 39 amino acids of bovine rhodopsin inframe with human and rodent T2R coding sequences (Mehta and Singh,Biotechniques 26:1082-1086 (1999). All receptors were cloned into apEAK10 mammalian expression vector (Edge Biosystems, MD). ModifiedHEK-293 cells (PEAK^(rapid) cells; Edge BioSystems, MD) were grown andmaintained at 37° C. in UltraCulture medium (Bio Whittaker) supplementedwith 5% fetal bovine serum, 100 μg/ml Gentamycin sulphate (Fisher), 1∥g/ml Amphotericin B and 2 mM GlutaMax I (Lifetechnologies). Fortransfection, cells were seeded onto matrigel coated 24-well cultureplates or 35 mm recording chambers. After 24 h at 37° C., cells werewashed in OptiMEM medium (Lifetechnologies) and transfected usingLipofectAMINE reagent (Lifetechnologies). Transfection efficiencies wereestimated by co-transfection of a GFP reporter plasmid, and weretypically >70%. Immunofluoresence staining, and activity assays wereperformed 36-48 h after transfection.

For immunostaining, transfected cells were grown on coated glasscoverslips, fixed for 20 min in ice-cold 2% paraformaldehyde, blockedwith 1% BSA, and incubated for 4-6 h at 4° C. in blocking buffercontaining a 1:1000 dilution of anti-rhodopsin mAb B6-30 (Hargrave, etal. Exp Eye Res 42:363-373 (1986)). Chimeric receptor expression wasvisualized using FITC-coupled donkey anti-mouse secondary antibodies(Jackson Immunochemical).

Two parallel strategies were employed to identify ligands for T2Rs. Inone, a random set of human, rat and mouse T2R receptors were selectedand individually tested against a collection of 55 bitter and sweettastants, including (shown with maximum concentrations tested): 5 mMaristolochic acid, 5 mM atropine, 5 mM brucine, 5 mM caffeic acid, 10 mMcaffeine, 1 mM chloroquine, 5 mM cycloheximide, 10 mM denatoniumbenzoate, 5 mM (−) epicatechin, 10 mM L-leucine, 10 mM L-lysine, 10 mMMgCl₂, 5 mM naringin, 10 mM nicotine, 2.5 mM papavarine hydrochloride, 3mM phenyl thiocarbamide, 10 mM 6-n-propyl thiouracil, 1 mM quinacrine, 1mM quinine hydrochloride, 800 μM raffinose undecaacetate, 3 mM salicin,5 mM sparteine, 5 mM strychnine nitrate, 3 mM sucrose octaacetate, 2 mMtetraethyl ammonium chloride, 10 mM L-tyrosine, 5 mM yohimbine, 10 mMeach of L-glycine, L-alanine, D-tryptophan, L-phenylalanine, L-arginine,sodium saccharin, aspartame, sodium cyclamate, acesulfame K, 150 mM eachof sucrose, lactose, maltose, D-glucose, D-fructose, D-galactose,D-sorbitol, 0.1% monellin, 0.1% thaumatin. Additional sweet tastantswere 150 μM alitame, 1.8 mM dulcin, 800 μM stevioside, 1.9 mMcyanosusan, 600 μM neohesperidin dihydrochalcone, 10 mM xylitol, 9.7 mMH-Asp-D-Ala-OTMCP, 70 μM N-Dmb-L-Asp-L-Phe-Ome, and 12 μMN-Dmb-L-Asp-D-Val-(S)-α methylbenzylamide. In these assays, functionalcoupling was assessed based on four criteria: tastant selectivity,temporal specificity, and receptor- and G protein-dependence. The secondstrategy relied upon data on the genetics of bitter perception in miceto link candidate receptors with specific tastants.

Nearly 30 years ago, it was first reported that various inbred strainsof mice differ in their sensitivity to the bitter compoundsucrose-octaacetate (Warren and Lewis, Nature 227:77-78(1970)).Subsequently, a number of studies demonstrated that this straindifference was due to allelic variation at a single genetic locus (Soa)(Whitney and Harder, Behav Genet 16:559-574 (1986); Capeless et al.,Behav Genet 22:655-663 (1992)). These findings were extended toadditional loci influencing sensitivity to various bitter tastants,including raffinose undecaacetate (Rua), cycloheximide (Cyx), copperglycinate (Glb), and quinine (Qui) (Lush, Genet. Res. 44:151-160 (1984);Lush, Genet. Res. 47:117-123 (1986), Lush and Holland, (1988)). Geneticmapping experiments showed that the Soa, Rua, Cyx, Qui and Glb loci areclustered at the distal end of chromosome 6 (Lush and Holland, Genet.Res. 52:207-212 (1988); Capeless et al., Behav Genet 22:655-663 (1992)).In view of the above-described localization of various T2R genes tobitter-associated loci in mice, T2R receptors from this array wereconstructed as corresponding rho-mT2R chimeras and individuallytransfected into HEK-293 cells expressing the promiscuous Gα15 protein.After loading the cells with FURA-2, responses to sucrose octaacetate,raffinose undecaacetate, copper glycinate, quinine, and cycloheximidewere assayed.

Transfected cells were washed once in Hank's balanced salt solution with1 mM sodium pyruvate and 10 mM HEPES, pH 7.4 (assay buffer), and loadedwith 2 μM FURA-2 AM (Molecular Probes) for 1 h at room temperature. Theloading solution was removed and cells were incubated in 200 μl of assaybuffer for 1 h to allow the cleavage of the AM ester. For mostexperiments, 24-well tissue culture plates containing cells expressing asingle rho-T2R were stimulated with 200 μl of a 2× tastant solution (seenext section). [Ca²⁺]i changes were monitored using a Nikon Diaphot 200microscope equipped with a 10×/0.5 fluor objective with the TILL imagingsystem (T.I.L.L Photonics GmbH). Acquisition and analysis of thefluorescence images used TILL-Vision software. Generally, [Ca²⁺]i wasmeasured for 80-120 s by sequentially illuminating cells for 200 ms at340 nm and 380 nm and monitoring the fluorescence emission at 510 nmusing a cooled CCD camera. The F₃₄₀/F₃₈₀ ratio was analyzed to measure[Ca²⁺]i.

Kinetics of activation and deactivation were measured using a bathperfusion system. Cells were seeded onto a 150 μl microperfusionchamber, and test solutions were pressure-ejected with a picospritzerapparatus (General Valve, Inc.). Flow-rate was adjusted to ensurecomplete exchange of the bath solution within 4-5 s. In the case ofmT2R5, the entire camera field was measured since >70% of the cellsresponded to cycloheximide. For mT2R8 and hT2R4, 100 areas of interestin each were averaged for each experiment.

Cells expressing mT2R5 specifically responded to cycloheximide (FIG. 3).The response occurred in nearly all transfected cells and was receptor-and Gα15-dependent because cells lacking either of these components didnot trigger [Ca2+]i changes, even at 5000-fold higher cycloheximideconcentration. As expected for this coupling system, the tastant-inducedincrease in [Ca2+]i was due to release from internal stores, sinceanalogous results were obtained in nominally zero [Ca2+]out. Theactivation of mT2R5 by cycloheximide is very selective, as this receptordid not respond to any other tastants, even at concentrations that farexceeded their biologically relevant range of action (Saroli,Naturwissenschaften 71:428-9 (1984); Glendinning, Behav Neurosci113:840-854 (1994))(FIG. 4 a,b). While cycloheximide is only moderatelybitter to humans, it is strongly aversive to rodents with a sensitivitythreshold of ˜0.25 μM (Kusano et al., Appl. Exptl. Zool. 6:40-50 (1971);Lush and Holland, Genet. Res. 52:207-212 (1988)). In the cell-basedassay described herein, the concentration of cycloheximide required toinduce half-maximal response of mT2R5 was 0.5 μM, and the threshold was˜0.2 μM (FIG. 4 c,d). Notably, this dose-response closely matches thesensitivity range of cycloheximide tasting in mice.

To examine the kinetics of the cycloheximide response, rho-mT2R5transfected cells were placed on a microperfusion chamber and superfusedwith test solutions under various conditions. The cells showed robusttransient responses to micromolar concentrations of cycloheximide thatclosely follow application of the stimulus (latency<1 s). As expected,when the tastant was removed, [Ca2+]i returned to baseline. A prolongedexposure to cycloheximide (>10 s) resulted in adaptation: a fastincrease of [Ca2+]i followed by a gradual, but incomplete decline to theresting level (FIG. 4 a). Similarly, successive applications ofcycloheximide led to significantly reduced responses, indicative ofdesensitization (Lefkowitz et al., Cold Spring Harb Symp Quant Biol57:127-133 (1992)). This is likely to occur at the level of thereceptor, since responses of a control, co-transfected mGluR1 were notaltered during the period of cycloheximide desensitization.

To determine whether other T2Rs are also activated by bitter compounds,11 rhodopsin-tagged human T2R receptors were assayed by individuallytransfecting them into HEK-293 cells expressing Gα15. Each transfectedline was tested against a battery of bitter and sweet tastants,including amino acids, peptides, and other natural and syntheticcompounds. These experiments demonstrated that the intensely bittertastant denatonium induced a significant transient increase in [Ca2+]iin cells transfected with one of the human candidate taste receptors,hT2R4, but not in control untransfected cells (FIG. 3), or in cellstransfected with other hT2Rs. The denatonium response had a strongdose-dependency with a threshold of ˜100 μM. Interestingly, hT2R4displayed a limited range of promiscuity since it also responded to highconcentrations of the bitter tastant 6-n-propyl-2-thiouracil (PROP)(FIG. 5).

If the responses of hT2R4 reflect the in vivo function of this receptor,it was hypothesized that similarly tuned receptors might be found inother species. The mouse receptor mT2R8 is a likely ortholog of hT2R4:they share ˜70% identity, while the next closest receptor is only 40%identical; these two genes are contained in homologous genomicintervals. A rho-mT2R8 chimeric receptor was generated and examined forits response to a wide range of tastants. Indeed, mT2R8, like its humancounterpart, is activated by denatonium and by high concentrations ofPROP (FIGS. 3 and 5). No other tastants elicited significant responsesfrom cells expressing mT2R8. Because these two receptors share only 70%identity, the similarity in their responses to bitter compounds atteststo their role as orthologous bitter taste receptors.

Example VIII Cycloheximide Non-Taster Mice have Mutations in the mT2R5Taste Receptor

The demonstration that mT2R5 functions as a high affinity receptor forcycloheximide suggested that the mT2R5 gene might correspond to the Cyxlocus. In situ hybridization to tissue sections demonstrated that theexpression profile of mT2R5 is indistinguishable between taster andnon-taster strains (FIG. 6). To determine the linkage between mT2R5 andthe Cyx locus, polymorphisms in the mT2R5 gene were identified and theirdistribution in a recombinant inbred panel from a C57BL/6J(non-taster)×DBA/2J (taster) cross was determined. Tight linkage wasfound between mT2R5 and the Cyx locus. To test the possibility thatmutations in the mT2R5 gene were responsible for the Cyx phenotype, themT2R5 gene was isolated from several additional well-characterizedcycloheximide taster (CBA/Ca, BALB/c, C3H/He) and non-taster (129/Sv)strains and their nucleotide sequences determined. Indeed, as would beexpected if mT2R5 functions as the cycloheximide receptor in thesestrains, all the tasters share the same mT2R5 allele as DBA/2J, whilethe non-tasters share the C57BL/6 allele, which carries missensemutations (FIG. 6), including 3 non-conservative amino acidsubstitutions (T44I, G155D and L294R).

If the mT2R5 C57BL/6 allele is responsible for the taste deficiency ofCyx mutants, its cycloheximide dose-response might recapitulate thesensitivity shift seen in Cyx mutant strains. Two-bottle preferencetests have shown that Cyx taster strains avoid cycloheximide with athreshold of 0.25 μM, while non-tasters have a ˜8-fold decrease insensitivity (e.g. they, are non-tasters at 1 μM, but strongly avoidcycloheximide at 8 μM). A rho-mT2R5 fusion was constructed with themT2R5 gene from a non-taster strain, and its dose response compared withthat of the receptor from taster strains. Remarkably, mT2R5 from thenon-taster strains displays a shift in cycloheximide sensitivity (FIG. 4d) that resembles the sensitivity of these strains to this bittertastant. Taken together, these results validate mT2R5 as a cycloheximidereceptor, and strongly suggest that mT2R5 corresponds to the Cyx locus.

Example IX T2Rs Couple to Gustducin

The above-described demonstration that T2Rs are co-expressed withgustducin suggests that T2Rs activate this G-protein in response tobitter tastants. To investigate the selectivity of T2R-G-proteincoupling, mT2R5 was chosen for study because its activation bycycloheximide recapitulates mouse taste responses. Rho-tagged mT2R5 andgustducin were prepared using a baculovirus expression system.mT2R5-containing membranes were incubated with various purifiedG-proteins, including gustducin, and measured tastant-induced GTP-γSbinding (Hoon et al., Biochem J 309:629-636 (1995)). Specifically,infectious Bacmid containing rhodopsin tagged mT2R5 (DBA/2-allele) wasproduced using the Bac-to-Bac system (Lifetechnologies, MD). Insectlarval cells were infected for 60 h with recombinant Bacmid andmembranes were prepared as described previously (Ryba and Tirindelli, JBiol Chem, 270:6757-6767 (1995)). Peripheral proteins were removed bytreatment with 8 M urea and membranes were resuspended in 10 mM HEPESpH7.5, 1 mM EDTA and 1 mM DTT. The expression of rho-mT2R5 was assessedby Western blot using mAb B6-30 and quantitated by comparison with knownamounts of rhodopsin. Approximately 300 pmol of rho-mT2R5 could beobtained from 2×10⁸ infected cells. Gustducin and Gβ₁γ₈ heterodimerswere isolated as described previously (Hoon et al., Biochem J309:629-636 (1995); Ryba and Tirindelli, J Biol Chem, 270:6757-6767(1995)). Receptor-catalyzed exchange of GDP for GTPγS on gustducin andother G-protein α-subunits was measured in the presence of 10 nMrho-mT2R5, 100 μM GDP, and 20 μM Gβ₁γ₈. All measurements were made at15-minute time points, and reflect the initial rate of GTPγS binding.

These GTP-γS binding assays revealed exquisite cycloheximide-dependentcoupling of mT2R5 to gustducin (FIG. 7). In contrast, no coupling wasseen with Gαs, Gαi, Gαq or Gαo. No significant GTPγS binding wasobserved in the absence of receptor, gustducin or βγ-heterodimers. Thehigh selectivity of T2R5 for gustducin, and the exclusive expression ofT2Rs in taste receptor cells that contain gustducin, affirm thehypothesis that T2Rs function as gustducin-linked taste receptors.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. An isolated nucleic acid encoding a taste transduction Gprotein-coupled receptor having G protein-coupled receptor activity, thereceptor comprising a polypeptide with greater than 95% amino acidsequence identity to SEQ ID NO:26, wherein said receptor detects bittertastants.
 2. The nucleic acid of claim 1, wherein the polypeptidesequence is SEQ ID NO:26.
 3. An isolated expression vector comprisingthe nucleic acid of claim
 1. 4. The isolated expression vector of claim3, wherein the polypeptide sequence is SEQ ID NO:26.
 5. An isolated cellcomprising the expression vector of claim
 3. 6. The isolated cell ofclaim 5, wherein said polypeptide sequence is SEQ ID NO:26.